Treatment of cone cell degeneration with transfected lineage negative hematopoietic stem cells

ABSTRACT

A method of preserving cone cells in the eye of a mammal suffering from a retinal degenerative disease comprises isolating from the bone marrow of the mammal a lineage negative hematopoietic stem cell population that includes endothelial progenitor cells, transfecting cells from the stem cell population with a gene that operably encodes an antiangiogenic fragment of human tryptophanyl tRNA synthetase (TrpRS), and subsequently intravitreally injecting the transfected cells into the eye of the mammal in an amount sufficient to inhibit the degeneration of cone cells in the retina of the eye. The treatment may be enhanced by stimulating proliferation of activated astrocytes in the retina using a laser.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application for patent Ser.No. 11/168,130, filed on Jun. 28, 2005, which is a continuation-in-partof U.S. application for patent Ser. No. 10/933,634, filed on Sep. 3,2004, which is a continuation-in-part of U.S. application for patentSer. No. 10/833,743, filed on Apr. 28, 2004, which is acontinuation-in-part of U.S. application for patent Ser. No. 10/628,783,filed on Jul. 25, 2003, which claims the benefit of ProvisionalApplication for Patent Ser. No. 60/398,522, filed on Jul. 25, 2002, andwhich claims the benefit of Provisional Application for Patent Ser. No.60/467,051, filed on May 2, 2003, each of which is incorporated hereinby reference.

STATEMENT OF GOVERNMENT INTEREST

A portion of the work described herein was supported by grant numberCA92577 from the National Cancer Institute and by grants number EY11254,EY12598 and EY125998 from the National Institutes of Health. The UnitedStates Government has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to mammalian stem cells. More particularly theinvention is related to treatment of cone cell degeneration byadministration to the eye of transfected lineage negative hematopoieticstem cell (Lin⁻HSC) populations derived from bone marrow.

BACKGROUND OF THE INVENTION

Age related macular degeneration (ARMD) and diabetic retinopathy (DR)are the leading causes of visual loss in industrialized nations and doso as a result of abnormal retinal neovascularization. Since the retinaconsists of well-defined layers of neuronal, glial, and vascularelements, relatively small disturbances such as those seen in vascularproliferation or edema can lead to significant loss of visual function.Inherited retinal degenerations, such as retinitis pigmentosa (RP), arealso associated with vascular abnormalities, such as arteriolarnarrowing and vascular atrophy. Most inherited human retinaldegenerations specifically affect rod photoreceptors, but there is alsoa concomitant loss of cones, the principal cellular component of themacula, which is the region of the retina in humans that is responsiblefor central, fine visual acuity. Cone-specific survival factors havebeen described recently (Mohand-Said et al. 1998, Proc. Natl. Acad. Sci.USA, 95: 8357-8362), which may facilitate cone survival in mouse modelsof retinal degeneration.

Inherited degenerations of the retina affect as many as 1 in 3500individuals and are characterized by progressive night blindness, visualfield loss, optic nerve atrophy, arteriolar attenuation, alteredvascular permeability, and central loss of vision often progressing tocomplete blindness (Heckenlively, J. R., editor, 1988; RetinitisPigmentosa, Philadelphia: JB Lippincott Co.). Molecular genetic analysisof these diseases has identified mutations in over 110 different genesaccounting for only a relatively small percentage of the known affectedindividuals (Humphries et al., 1992, Science 256:804-808; Farrar et al.2002, EMBO J. 21:857-864.). Many of these mutations are associated withenzymatic and structural components of the phototransduction machineryincluding rhodopsin, cGMP phosphodiesterase, rds peripherin, and RPE65.Despite these observations, there are still no effective treatments toslow or reverse the progression of these retinal degenerative diseases.Recent advances in gene therapy have led to successful reversal of therds (Ali et al. 2000, Nat. Genet. 25:306-310) and rd (Takahashi et al.1999, J. Virol. 73:7812-7816) phenotypes in mice and the RPE65 phenotypein dogs (Acland et al. 2001, Nat. Genet. 28:92-95) when the wild typetransgene is delivered to photoreceptors or the retinal pigmentedepithelium (RPE) in animals with a specific mutation.

For many years it has been known that a population of stem cells existsin the normal adult circulation and bone marrow. Differentsub-populations of these cells can differentiate along hematopoieticlineage positive (Lin⁺) or lineage negative (Lin⁻) lineages.Furthermore, the lineage negative hematopoietic stem cell (HSC)population has recently been shown to contain endothelial progenitorcells (EPC) capable of forming blood vessels in vitro and in vivo (SeeAsahara et al. 1997, Science 275: 964-7). These cells can participate innormal and pathological postnatal angiogenesis (See Lyden et al. 2001Nat. Med. 7, 1194-201; Kalka et al. 2000, Proc. Natl. Acad. Sci. U.S.A.97:3422-7; and Kocher et al. 2001, Nat. Med. 7: 430-6) as well asdifferentiate into a variety of non-endothelial cell types includinghepatocytes (See Lagasse et al. 2000, Nat. Med. 6:1229-34), microglia(See Priller et al. 2002 Nat. Med. 7:1356-61), cardiomyocytes (See Orlicet al. 2001, Proc. Natl. Acad. Sci. U.S.A. 98:10344-9), and epithelium(See Lyden et al. 2001, Nat. Med. 7:1194-1201). Although these cellshave been used in several experimental models of angiogenesis, themechanism of EPC targeting to neovasculature is not known and nostrategy has been identified that will effectively increase the numberof cells that contribute to a particular vasculature.

Hematopoietic stem cells from bone marrow are currently the only type ofstem cell commonly used for therapeutic applications. Bone marrow HSC'shave been used in transplants for over 40 years. Currently, advancedmethods of harvesting purified stem cells are being investigated todevelop therapies for treatment of leukemia, lymphoma, and inheritedblood disorders. Clinical applications of stem cells in humans have beeninvestigated for the treatment of diabetes and advanced kidney cancer inlimited numbers of human patients.

SUMMARY OF THE INVENTION

The present invention provides a method of ameliorating cone celldegeneration in the retina of a mammal that suffers from an oculardisease. The method comprises the step of administering to the retina ofthe mammal a transfected, mammalian bone marrow-derived, lineagenegative hematopoietic stem cell population, which compriseshematopoietic stem cells and endothelial progenitor cells, in which thecells also express an antiangiogenic fragment of tryptophanyl tRNAsythetase (TrpRS). The cells are administered in an amount sufficient toretard cone cell degeneration in the retina. Preferably, the transfectedstem cell population expresses a fragment of TrpRS selected from thegroup consisting of T2-TrpRS (SEQ ID NO: 3, FIG. 26), T2-TrpRS-GD (SEQID NO: 4; FIG. 26), mini-TrpRS (SEQ ID NO: 5; (FIG. 27), and T1-TrpRS(SEQ ID NO: 6; FIG. 27).

A preferred method comprises isolating from the bone marrow of a mammalsuffering from an ocular disease a lineage negative hematopoietic stemcell population that includes endothelial progenitor cells, transfectingthe isolated cells with a gene that operably encodes an antiangiogenicfragment of TrpRS, and subsequently intravitreally injecting theisolated stem cells into an eye of the mammal in a number sufficient toameliorate the degeneration of cone cells in the retina.

The methods of the present invention utilize an isolated, mammalian,lineage negative hematopoietic stem cell (Lin⁻HSC) population (i.e.,hematopoietic stem cells (HSCs) that do not express lineage surfaceantigens (Lin) on their cell surface) derived from mammalian bonemarrow. Preferably, the cells are autologous stem cells (i.e., derivedfrom the bone marrow of the individual mammal that is to be treated).The isolated, mammalian, population of Lin⁻HSCs, includes endothelialprogenitor cells (EPC), also known as endothelial precursor cells, thatselectively target activated retinal astrocytes when intravitreallyinjected into the eye. Preferably the mammal is a human.

In a preferred embodiment the Lin⁻HSC populations of the presentinvention are isolated by extracting bone marrow from a mammal sufferingfrom an ocular disease; separating a plurality of monocytes from thebone marrow; labeling the monocytes with biotin-conjugated lineage panelantibodies to one or more lineage surface antigens, removing monocytesthat are positive for the lineage surface antigens, and then recoveringa Lin⁻HSC population containing EPCs. Preferably the monocytes arelabeled with biotin-conjugated lineage panel antibodies to one or morelineage surface antigen selected from the group consisting of CD2, CD3,CD4, CD11, CD11a, Mac-1, CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45,Ly-6G, TER-119, CD45RA, CD56, CD64, CD68, CD86, CD66b, HLA-DR, andCD235a (Glycophorin A). Preferably, at least about 20% of the cells ofthe isolated Lin⁻HSC population of the present invention express thesurface antigen CD31. The isolated cells are then transfected with agene that operably encodes an antiangiogenic TrpRS fragment, andsubsequently administered to the diseased eye of the mammal, preferablyby intraocular injection. In a preferred embodiment, at least about 50%the isolated Lin⁻HSCs express the surface antigen CD31 and at leastabout 50% the isolated Lin⁻HSCs express the surface antigen CD117(c-kit).

The EPC's within the population of Lin⁻HSCs of the present inventionextensively incorporate into developing retinal blood vessels and intothe neuronal network of the retina, and remain stably incorporated intoneovasculature and neuronal network of the eye. The normal mouse retinais predominantly rods; however, in mice treated by the methods of thepresent invention, the rescued cells after treatment with Lin-HSCs weresurprisingly nearly all cones.

In one preferred embodiment, the cells of the isolated Lin⁻HSCpopulations are also transfected with a gene encoding a therapeuticallyuseful protein in addition to the antiangiogenic TrpRS fragment. Forexample, the cells can be transfected with polynucleotides that operablyencode for a neurotrophic agent or another antiangiogenic agent thatselectively targets neovasculature and inhibits new vessel formationwithout affecting already established vessels through a form ofcell-based gene therapy. In one embodiment, the Lin⁻HSC populationsuseful in the methods of the present invention include a gene encodingan angiogenesis inhibiting peptide. The angiogenesis inhibiting Lin⁻HSCsare useful for modulating abnormal blood vessel growth in diseases suchas ARMD, DR and certain retinal degenerations associated with abnormalvasculature. In another preferred embodiment, the transfected, Lin⁻HSCsof the present invention include a gene encoding a neurotrophic peptide.The neurotrophic Lin⁻HSCs are useful for promoting neuronal rescue inocular diseases involving retinal neural degeneration, such as glaucoma,retinitis pigmentosa, and the like.

A particular advantage of ocular treatments with the transfected Lin⁻HSCpopulations of the present invention is a vasculotrophic andneurotrophic rescue effect observed in eyes intravitreally treated withthe Lin⁻HSCs. Retinal neurons and photoreceptors, particularly cones,are preserved and some measure of visual function can be maintained ineyes treated with the transfected Lin⁻HSCs of the invention.

Preferably, the diseased retina to be treated by the methods of theinvention includes activated astrocytes. This can be accomplished byearly treatment of the eye when there is an associated gliosis, or byusing a laser to stimulate local proliferation of activated astrocytes.

BRIEF DESCRIPTION OF THE DRAWINGS

In the DRAWINGS:

FIG. 1 depicts schematic diagrams of developing mouse retina. (a)Development of primary plexus. (b) The second phase of retinal vesselformation. GCL, ganglion cell layer; IPL, inner plexus layer; INL, innernuclear layer; OPL, outer plexus layer; ONL, outer nuclear layer; RPE,retinal pigment epithelium; ON, optic nerve; P, periphery. Panel (c)depicts flow cytometric characterization of bone marrow-derived Lin⁺HSCand Lin⁻HSC separated cells. Top row: Dot plot distribution ofnon-antibody labeled cells, in which R1 defines the quantifiable-gatedarea of positive PE-staining; R2 indicates GFP-positive; Middle row:Lin⁻HSC(C57B/6) and Bottom row: Lin⁺HSC(C57B/6) cells, each cell linelabeled with the PE-conjugated antibodies for Sca-1, c-kit, Flk-1/KDR,CD31. Tie-2 data was obtained from Tie-2-GFP mice. Percentages indicatepercent of positive-labeled cells out of total Lin⁻HSC or Lin⁺HSCpopulation.

FIG. 2 depicts engraftment of Lin⁻HSCs into developing mouse retina. (a)At four days post-injection (P6) intravitreally injected eGFP⁺Lin⁻HSCcells attach and differentiate on the retina (b) Lin⁻HSC(B6.129S7-Gtrosa26 mice, stained with β-gal antibody) establishthemselves ahead of the vasculature stained with collagen IV antibody(asterisk indicates tip of vasculature). (c) Most of Lin⁺HSC cells(eGFP⁺) at four days post-injection (P6) were unable to differentiate.(d) Mesenteric eGFP⁺ murine EC four days post-injection (P6). (e) Lin⁻HSCs (eGFP⁺) injected into adult mouse eyes. (f) Low magnification ofeGFP⁺Lin⁻ HSCs (arrows) homing to and differentiating along thepre-existing astrocytic template in the GFAP-GFP transgenic mouse. (g)Higher magnification of association between Lin⁻ cells (eGFP) andunderlying astrocyte (arrows). (h) Non-injected GFAP-GFP transgeniccontrol. (i) Four days post-injection (P6), eGFP⁺Lin⁻HSCs migrate to andundergo differentiation in the area of the future deep plexus. Leftfigure captures Lin⁻ HSC activity in a whole mounted retina; rightfigure indicates location of the Lin⁻ cells (arrows) in the retina (topis vitreal side, bottom is scleral side). (j) Double labeling withα-CD31-PE and α-GFP-alexa 488 antibodies. Seven days after injection,the injected Lin⁻HSCs (eGFP), red) were incorporated into thevasculature (CD31). Arrowheads indicate the incorporated areas. (k)eGFP⁺Lin⁻ HSC cells form vessels fourteen days post-injection (P17). (land m) Intra-cardiac injection of rhodamine-dextran indicates that thevessels are intact and functional in both the primary (l) and deepplexus (m).

FIG. 3 shows that eGFP⁺Lin⁻HSC cells home to the gliosis (indicated byGFAP expressing-astrocytes, far left image) induced by both laser (a)and mechanical (b) induced injury in the adult retina (asteriskindicates injured site). Far right images are a higher magnification,demonstrating the close association of the Lin⁻HSCs and astrocytes.Calibration bar=20 μM.

FIG. 4 shows that Lin⁻HSC cells rescue the vasculature of the retinaldegeneration mouse. (a-d) Retinas at 27 days post-injection (P33) withcollagen IV staining; (a) and (b), retinas injected with Lin⁺ HSC cells(Balb/c) showed no difference in vasculature from normal FVB mice; (c)and (d) retinas injected with Lin⁻HSCs (Balb/c) exhibited a richvascular network analogous to a wild-type mouse; (a) and (c), frozensections of whole retina (top is vitreal side, bottom is scleral side)with DAPI staining; (b) and (d), deep plexus of retinal whole amount;(e) bar graph illustrating the increase in vascularity of the deepvascular plexus formed in the Lin⁻HSC cell-injected retinas (n=6). Theextent of deep retinal vascularization was quantified by calculating thetotal length of vessels within each image. Average total length ofvessels/high power field (in microns) for Lin⁻HSC, Lin⁺HSC or controlretinas were compared. (f) Comparison of the length of deep vascularplexus after injection with Lin⁻HSC(R, right eye) or Lin⁺HSC (L, lefteye) cells from rd/rd mouse. The results of six independent mice areshown (each color represents each mouse). (g) and (h) Lin⁻HSC cells also(Balb/c) rescued the rd/rd vasculature when injected into P15 eyes. Theintermediate and deep vascular plexus of Lin⁻HSC (G) or Lin⁺HSC(H) cellinjected retinas (one month after injection) are shown.

FIG. 5 depicts photomicrographs of mouse retinal tissue: (a) deep layerof retinal whole mount (rd/rd mouse), five days post-injection (P11)with eGFP⁺Lin⁻HSCs visible (gray). (b) and (c) P60 retinal vasculatureof Tie-2-GFP (rd/rd) mice that received Balb/c Lin⁻ cells (b) or Lin⁺HSCcell (c) injection at P6. Only endogenous endothelial cells(GFP-stained) are visible in the left panels of (b) and (c). The middlepanels of (b) and (c) are stained with CD31 antibody; arrows indicatethe vessels stained with CD31 but not with GFP, the right panels of (b)and (c) show staining with both GFP and CD31. (d) α-SMA staining ofLin⁻HSC injected (left panel) and control retina (right panel).

FIG. 6 shows that T2-TrpRS-transfected Lin⁻HSCs inhibit the developmentof mouse retinal vasculature. (a) Schematic representation of humanTrpRS, T2-TrpRS and T2-TrpRS with an Igk signal sequence at the aminoterminus. (b) T2-TrpRS transfected Lin⁻HSC-injected retinas expressT2-TrpRS protein in vivo. (1) Recombinant T2-TrpRS produced in E. coli;(2) Recombinant T2-TrpRS produced in E. coli; (3) Recombinant T2-TrpRSproduced in E. coli; (4) control retina; (5) Lin⁻HSC+pSecTag2A (vectoronly) injected retina; (6) Lin⁻HSC+pKLe135 (Igk-T2-TrpRS in pSecTag)injected retina. (c-f) Representative primary (superficial) andsecondary (deep) plexuses of injected retinas, seven dayspost-injection; (c) and (d) Eyes injected with empty plasmid-transfectedLin⁻HSC developed normally; (e) and (f) the majority ofT2-TrpRS-transfected Lin⁻HSC injected eyes exhibited inhibition of deepplexus; (c) and (e) primary (superficial) plexus; (d) and (f) secondary(deep) plexus). Faint outline of vessels observed in (f) are“bleed-through” images of primary network vessels shown in (e).

FIG. 7 shows the DNA sequence encoding His₆-tagged T2-TrpRS, SEQ ID NO:1.

FIG. 8 shows the amino acid sequence of His₆-tagged T2-TrpRS, SEQ ID NO:2.

FIG. 9 illustrates photomicrographs and electroretinograms (ERG) ofretinas from mice whose eyes were injected with the Lin⁻HSC of thepresent invention and with Lin⁺HSC (controls).

FIG. 10 depicts statistical plots showing a correlation between neuronalrescue (y-axis) and vascular rescue (x-axis) for both the intermediate(Int.) and deep vascular layers of rd/rd mouse eyes treated withLin⁻HSC.

FIG. 11 depicts statistical plots showing no correlation betweenneuronal rescue (y-axis) and vascular rescue (x-axis) for rd/rd mouseeyes that were treated with Lin⁺HSC.

FIG. 12 is a bar graph of vascular length (y-axis) in arbitrary relativeunits for rd/rd mouse eyes treated with the Lin HSC (dark bars) anduntreated (light bars) rd/rd mouse eyes at time points of 1 month (1M),2 months (2M), and 6 months (6M) post-injection.

FIG. 13 includes three bar graphs of the number of nuclei in the outerneural layer (ONR) of rd/rd mice at 1 month (1M), 2 months (2M) and 6months (6M), post-injection, and demonstrates a significant increase inthe number of nuclei for eyes treated with Lin⁻HSC (dark bars) relativeto control eyes treated with Lin⁺HSC (light bars).

FIG. 14 depicts plots of the number of nuclei in the outer neural layerfor individual rd/rd mice, comparing the right eye (R, treated withLin⁻HSC) relative to the left eye (L, control eye treated with Lin⁺HSC)at time points (post injection) of 1 month (1M), 2 months (2M), and 6months (6M); each line in a given plot compares the eyes of anindividual mouse.

FIG. 15 depicts retinal vasculature and neural cell changes in rd1/rd1(C3H/HeJ, left panels) or wild type mice (C57BL/6, right panels).Retinal vasculature of intermediate (upper panels) or deep (middlepanels) vascular plexuses in whole-mounted retinas (red: collagen IV,green: CD31) and sections (red: DAPI, green: CD31, lower panels) of thesame retinas are shown (P: postnatal day). (GCL: ganglion cell layer,INL: inter nuclear layer, ONL: outer nuclear layer).

FIG. 16 shows that Lin⁻HSC injection rescues the degeneration of neuralcells in rd1/rd1 mice. (A, B and C), retinal vasculature of intermediate(int.) or deep plexus and sections of Lin⁻HSC injected eye (rightpanels) and contralateral control cell (CD31⁻) injected eye (leftpanels) at P30 (A), P60 (B), and P180 (C). (D), the average total lengthof vasculature (+ or − standard error of the mean) in Lin⁻HSC injectedor control cell (CD31⁻) injected retinas at P30 (left, n=10), P60(middle, n=10), and P180 (right, n=6). Data of intermediate (Int.) anddeep vascular plexus are shown separately (Y axis: relative length ofvasculature). (E), the average numbers of cell nuclei in the ONL at P30(left, n=10), P60 (middle, n=10), or P180 (right, n=6) of control cell(CD31⁻) or Lin⁻HSC injected retinas (Y axis: relative number of cellnuclei in the ONL). (F), Linear correlations between the length ofvasculature (X axis) and the number of cell nuclei in the ONL (Y axis)at P30 (left), P60 (middle), and P180 (right) of Lin⁻HSC or control cellinjected retinas.

FIG. 17 demonstrates that retinal function is rescued by Lin⁻HSCinjection. Electroretinographic (ERG) recordings were used to measurethe function of Lin⁻HSC or control cell (CD31⁻) injected retinas. (A andB), Representative cases of rescued and non-rescued retinas 2 monthsafter injection. Retinal section of Lin⁻HSC injected right eye (A) andCD31⁻ control cell injected left eye (B) of the same animal are shown(green: CD31 stained vasculature, red: DAPI stained nuclei). (C), ERGresults from the same animal shown in (A and B).

FIG. 18 shows that a population of human bone marrow cells can rescuedegenerating retinas in the rd1 mouse (A-C). The rescue is also observedin another model of retinal degeneration, rd10 (D-K). A, human Lin⁻HSCs(hLin⁻HSCs) labeled with green dye can differentiate into retinalvascular cells after intravitreal injection into C3SnSmn.CB17-Prkdc SCIDmice. (B and C), Retinal vasculature (left panels; upper: intermediateplexus, lower: deep plexus) and neural cells (right panel) in hlin⁻HSCinjected eye (B) or contralateral control eye (C) 1.5 months afterinjection. (D-K), Rescue of rd10 mice by Lin⁻HSCs (injected at P6).Representative retinas at P21 (D: Lin⁻HSCs, H: control cells), P30 (E:Lin⁻HSCs, I: control cells), P60 (F: Lin⁻HSCs, J: control cells), andP105 (G: Lin⁻HSCs, K: control cells) are shown (treated and control eyesare from the same animal at each time point). Retinal vasculature (upperimage in each panel is the intermediate plexus; the middle image in eachpanel is the deep plexus) was stained with CD31 (green) and Collagen IV(red). The lower image in each panel shows a cross section made from thesame retina (red: DAPI, green: CD31).

FIG. 19 demonstrates that crystallin αA is up regulated in rescued outernuclear layer cells after treatment with Lin-HSCs but not incontralateral eyes treated with control cells. Left panel; IgG controlin rescued retina, Middle panel; crystallin αA in rescued retina, Rightpanel; crystallin αA in non-rescued retina.

FIG. 20 includes tables of genes that are upregulated in murine retinasthat have been treated with the Lin⁻HSCs of the present invention. (A)Genes whose expression is increased 3-fold in mouse retinas treated withmurine Lin⁻HSCs. (B) Crystallin genes that are upregulated in mouseretinas treated with murine Lin⁻HSC. (C) Genes whose expression isincreased 2-fold in mouse retinas treated with human Lin⁻HSCs. (D) Genesfor neurotrophic factors or growth factors whose expression isupregulated in mouse retinas treated with human Lin HSCs.

FIG. 21 illustrates the distribution of CD31 and integrin α₆ surfaceantigens on CD133 positive (DC133⁺) and CD133 negative (CD133⁻) humanLin⁻HSC populations of the present invention. The left panels show flowcytometry scatter plots. The center and right panels are histogramsshowing the level of specific antibody expression on the cellpopulation. The Y axis represents the number of events and the X axisshows the intensity of the signal. A filled histogram shifted to theright of the outlined (control) histogram represents an increasedfluorescent signal and expression of the antibody above backgroundlevel.

FIG. 22 illustrates postnatal retinal development in wild-type C57/B16mice raised in normal oxygen levels (normoxia), at post natal days P0through P30.

FIG. 23 illustrates oxygen-induced retinopathy model in C57/B16 miceraised in high oxygen levels (hyperoxia; 75% oxygen) between P7 and P12,followed by normoxia from P12-P17.

FIG. 24 demonstrates vascular rescue by treatment with the Lin⁻HSCpopulations of the present invention in the oxygen-induced retinopathymodel.

FIG. 25 shows rescued photoreceptors in rd1 mouse outer nuclear layer(ONL) following intravitreal injection of Lin-HSC are predominantlycones. A small percentage of photoreceptors in the wild type mouseretina (upper panel) were cones as evidenced by expression of red/greencone opsin (A) while most cells of the ONL were positive for rodspecific rhodopsin (B). Retinal vasculature autofluoresces withpre-immune serum (C) but nuclear layers were completely negative forstaining with rod or cone-specific opsins. Rd/rd mouse retinas (lowerpanels) had a diminished inner nuclear layer and a nearly completelyatrophic ONL, both of which were negative for cone (D) or rod (G) opsin.Control, CD31-HSC treated eyes are identical to non-injected rd/rdretinas, without any staining for cone (E) or rod (H) opsin. Lin-HSCtreated contralateral eyes exhibited a markedly reduced, but clearlypresent ONL that is predominantly comprised of cones, as evidenced bypositive immunoreactivity for cone red/green opsin (F). A small numberof rods were also observed (I).

FIG. 26 shows the amino acid residue sequence of T2-TrpRS (SEQ ID NO: 3)and T2-TrpRS-GD (SEQ ID NO: 4)

FIG. 27 shows the amino acid residue sequence of mini-TrpRS (SEQ ID NO:5) and T1-TrpRS (SEQ ID NO: 6).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Stem cells are typically identified by the distribution of antigens onthe surface of the cells (for a detailed discussion see Stem Cells:Scientific Progress and Future Directions, a report prepared by theNational Institutes of Health, Office of Science Policy, June 2001,Appendix E: Stem Cell Markers, which is incorporated herein by referenceto the extent pertinent).

The present invention provides a method of ameliorating cone celldegeneration in the retina of a mammal that suffers from an oculardisease. A mammalian bone marrow-derived, isolated, lineage negativehematopoietic stem cell population, which comprises hematopoietic stemcells and endothelial progenitor cells, is transfected with a gene thatoperably encodes an antiangiogenic fragment of TrpRS, and is thenadministered to the retina of the mammal, preferably by intravitrealinjection. The cells are administered in an amount sufficient toameliorate cone cell degeneration in the retina.

A preferred method comprises isolating the lineage negative,hematopoietic stem cell population from the bone marrow of the mammal tobe treated, transfecting the isolated cells with a gene that operablyencodes an antiangiogenic fragment of TrpRS, and then administering thetransfected cells to the mammal in a number sufficient to ameliorate thedegeneration of cone cells in the retina. The cells can be obtained fromthe diseased mammal, preferably at an early stage of the ocular disease.Alternatively, the cells can be obtained prior to the onset of diseasein a patient known to have a genetic predisposition to an ocular diseasesuch as retinitis pigmentosa, for example. The cells can be stored untilneeded, and can then be transfected and injected prophylactically at thefirst observed indication of disease onset. Alternatively, the cells canbe transfected before storage. Preferably, the diseased retina includesactivated astrocytes, to which the stem cells are targeted. Accordingly,early treatment of the eye when there is an associated gliosis isbeneficial. Alternatively, the retina can be treated with a laser tostimulate local proliferation of activated astrocytes in the retinaprior to administering the autologous stem cells.

Hematopoietic stem cells are stem cells that are capable of developinginto various blood cell types e.g., B cells, T cells, granulocytes,platelets, and erythrocytes. The lineage surface antigens are a group ofcell-surface proteins that are markers of mature blood cell lineages,including CD2, CD3, CD11, CD11a, Mac-1 (CD11b:CD18), CD14, CD16, CD19,CD24, CD33, CD36, CD38, CD45, CD45RA, murine Ly-6G, murine TER-119,CD56, CD64, CD68, CD86 (B7.2), CD66b, human leucocyte antigenDR(HLA-DR), and CD235a (Glycophorin A). Hematopoietic stem cells that donot express significant levels of these antigens are commonly referredto a lineage negative (Lin⁻). Human hematopoietic stem cells commonlyexpress other surface antigens such as CD31, CD34, CD117 (c-kit) and/orCD133. Murine hematopoietic stem cells commonly express other surfaceantigens such as CD34, CD117 (c-kit), Thy-1, and/or Sca-1.

The present invention provides isolated hematopoietic stem cells that donot express significant levels of a “lineage surface antigen” (Lin) ontheir cell surfaces. Such cells are referred to herein as “lineagenegative” or “Lin⁻” hematopoietic stem cells. In particular, thisinvention provides a population of Lin⁻ hematopoietic stems cells(Lin⁻HSCs) that include endothelial progenitor cells (EPCs), which arecapable of incorporating into developing vasculature and thendifferentiating to become vascular endothelial cells. The cells aretransfected with a gene that operably encodes an antiangiogenic fragmentof TrpRS. Preferably, the transfected Lin⁻ HSC populations are presentin a culture medium such as phosphate buffered saline (PBS).

As used herein and in the appended claims, the phrase “adult” inreference to bone marrow, includes any bone marrow isolated postnatally,i.e., from juvenile and adult individuals, as opposed to embryos. Theterm “adult mammal” refers to all post natal individuals, i.e., bothjuvenile and fully mature mammals, as opposed to embryos.

The isolated, mammalian, lineage negative hematopoietic stem cell(Lin⁻HSC) populations of the invention include endothelial progenitorcells (EPCs). The isolated Lin⁻HSC populations preferably comprisemammalian cells in which at least about 20% of the cells express thesurface antigen CD31, which is commonly present on endothelial cells. Inother embodiment, at least about 50% of the cells express CD31, morepreferably at least about 65%, most preferably at least about 75%.Preferably at least about 50% of the cells of the Lin⁻HSC populations ofthe present invention preferably express the integrin α₆ antigen.

In one preferred murine Lin⁻HSC population embodiment, at least about50% of the cells express CD31 antigen and at least about 50% of thecells express the CD117 (c-kit) antigen. Preferably, at least about 75%of the Lin⁻HSC cells express the surface antigen CD31, more preferablyabout 81% of the cells. In another preferred murine embodiment, at leastabout 65% of the cells express the surface antigen CD 117, morepreferably about 70% of the cells. A particularly preferred embodimentof the present invention is a population of murine Lin⁻HSCs in whichabout 50% to about 85% of the cells express the surface antigen CD31 andabout 70% to about 75% of the cells express the surface antigen CD 117.

Another preferred embodiment comprises a human Lin⁻HSC population inwhich the cells are CD133 negative, in which at least about 50% of thecells express the CD31 surface antigen and at least about 50% of thecells express the integrin α₆ antigen. Yet another preferred embodimentcomprises a human Lin⁻HSC population in which the cells are CD133positive, in which at less than about 30% of the cells express the CD31surface antigen and less than about 30% of the cells express theintegrin α₆ antigen.

The isolated Lin⁻HSC populations of the present invention selectivelytarget astrocytes and incorporate into the retinal neovasculature whenintravitreally injected into the eye of the mammalian species, such as amouse or a human, from which the cells were isolated.

The isolated Lin⁻HSC populations of the present invention includeendothelial progenitor cells that differentiate to endothelial cells andgenerate vascular structures within the retina. In particular, theLin⁻HSC populations of the present invention are useful for thetreatment of retinal neovascular and retinal vascular degenerativediseases, and for repair of retinal vascular injury. The Lin⁻HSC cellsof the present invention also promote neuronal rescue in the retinapromote upregulation of anti-apoptotic genes. It has surprisingly beenfound that adult human Lin⁻HSC cells of the present invention caninhibit retinal degeneration even in severe combined immunodeficient(SCID) mice suffering from retinal degeneration. The normal mouse retinais predominantly rods, but the rescued cells, after treatment withLin-HSC are nearly all cones. Additionally, the Lin⁻HSC populations canbe utilized to treat retinal defects in the eyes of neonatal mammals,such as mammals suffering from oxygen induced retinopathy or retinopathyof prematurity.

The present invention also provides a method of treating ocular diseasesin a mammal comprising isolating from the bone marrow of the mammal alineage negative hematopoietic stem cell population that includesendothelial progenitor cells, transfecting the cells with a gene thatoperably encodes an antiangiogenic TrpRS fragment, and thenintravitreally injecting the isolated stem cells into an eye of themammal in a number sufficient to arrest the disease. The present methodcan be utilized to treat ocular diseases such as retinal degenerativediseases, retinal vascular degenerative diseases, ischemicretinopathies, vascular hemorrhages, vascular leakage, andchoroidopathies in neonatal, juvenile or fully mature mammals. Examplesof such diseases include age related macular degeneration (ARMD),diabetic retinopathy (DR), presumed ocular histoplasmosis (POHS),retinopathy of prematurity (ROP), sickle cell anemia, and retinitispigmentosa, as well as retinal injuries.

The number of stem cells injected into the eye is sufficient forarresting the disease state of the eye. For example, the number of cellscan be effective for repairing retinal damage of the eye, stabilizingretinal neovasculature, maturing retinal neovasculature, and preventingor repairing vascular leakage and vascular hemorrhage.

Cells of the Lin⁻HSC populations of the present invention aretransfected with a gene that operably encodes an antiangiogenic fragmentof TrpRS, e.g., the T1 and T2 fragments of TrpRS, which are described indetail in co-owned, co-pending U.S. patent application Ser. No.10/080,839, the disclosure of which is incorporated herein by reference.The transfected cells express the TrpRS fragment at a therapeuticallyeffective level when administered to the eye, e.g., by intravitrealinjection.

The transfected cells also can include any other gene which istherapeutically useful for treatment of retinal disorders. In onepreferred embodiment, the transfected Lin⁻HSCs of the present inventioninclude a gene operably encoding an antiangiogenic peptide in additionto the TrpRS fragment, including other antiangiogenic proteins, orprotein fragments. The transfected Lin⁻HSCs encoding an additionalantiangiogenic peptide are useful for treatment of retinal diseasesinvolving abnormal vascular development, such as diabetic retinopathy,and like diseases. Preferably the Lin⁻HSCs are human cells.

In another preferred embodiment, the transfected Lin⁻HSCs of the presentinvention also include a gene operably encoding a neurotrophic agentsuch as nerve growth factor, neurotrophin-3, neurotrophin-4,neurotrophin-5, ciliary neurotrophic factor, retinal pigmentedepithelium-derived neurotrophic factor, insulin-like growth factor,glial cell line-derived neurotrophic factor, brain-derived neurotrophicfactor, and the like. Such neurotrophic Lin⁻HSCs are useful forpromoting neuronal rescue in retinal neuronal degenerative diseases suchas glaucoma and retinitis pigmentosa, in treatment of injuries to theretinal nerves, and the like. Implants of ciliary neurotrophic factorhave been reported as useful for the treatment of retinitis pigmentosa(see Kirby et al. 2001, Mol. Ther. 3(2):241-8; Farrar et al. 2002, EMBOJournal 21:857-864). Brain-derived neurotrophic factor reportedlymodulates growth associated genes in injured retinal ganglia (seeFournier, et al., 1997, J. Neurosci. Res. 47:561-572). Glial cell linederived neurotrophic factor reportedly delays photoreceptor degenerationin retinitis pigmentosa (see McGee et al. 2001, Mol. Ther. 4(6):622-9).

The present invention also provides a method of isolating a lineagenegative hematopoietic stem cell population comprising endothelialprogenitor cells from bone marrow of a mammal. The method entails thesteps of (a) extracting bone marrow from an adult mammal; (b) separatinga plurality of monocytes from the bone marrow; (c) labeling themonocytes with biotin-conjugated lineage panel antibodies to one or morelineage surface antigens, preferably lineage surface antigens selectedfrom the group consisting of CD2, CD3, CD4, CD11, CD11a, Mac-1, CD14,CD16, CD19, CD24, CD33, CD36, CD38, CD45, Ly-6G (murine), TER-119(murine), CD45RA, CD56, CD64, CD68, CD86 (B7.2), CD66b, human leucocyteantigen DR(HLA-DR), and CD235a (Glycophorin A); (d) removing monocytesthat are positive for said one or more lineage surface antigens from theplurality of monocytes and recovering a population of lineage negativehematopoietic stem cells containing endothelial progenitor cells,preferably in which at least about 20% of the cells express CD31.

When the Lin⁻HSC are isolated from adult human bone marrow, preferablythe monocytes are labeled with biotin-conjugated lineage panelantibodies to lineage surface antigens such as CD2, CD3, CD4, CD11a,Mac-1, CD14, CD16, CD19, CD33, CD38, CD45RA, CD64, CD68, CD86 (B7.2),and CD235a. When the Lin⁻HSC are isolated from adult murine bone marrow,preferably the monocytes are labeled with biotin-conjugated lineagepanel antibodies to lineage surface antigens CD3, CD11, CD45, Ly-6G, andTER-119.

In a preferred method, the cells are isolated from adult human bonemarrow and are further separated by CD133 lineage. One preferred methodof isolating human Lin⁻HSCs includes the additional steps of labelingthe monocytes with a biotin-conjugated CD133 antibody and recovering aCD133 positive, Lin⁻HSC population. Typically, less than about 30% ofsuch cells express CD31 and less than about 30% of such cell expressintegrin α6. The human Cd133 positive, Lin⁻HSC populations of thepresent invention can target sites of peripheral ischemia-drivenneovascularization when injected into eyes that are not undergoingangiogenesis.

Another preferred method of isolating human Lin⁻HSCs includes theadditional steps of labeling the monocytes with a biotin-conjugatedCD133 antibody, removing CD133 positive cells, and recovering a CD133negative, Lin⁻HSC population. Typically, at least about 50% of suchcells express CD31 and at least about 50% of such cell express integrinα₆. The human CD133 negative, Lin⁻HSC populations of the presentinvention can incorporate into developing vasculature when injected intoeyes that are undergoing angiogenesis.

The present invention also provides methods for treating ocularangiogenic diseases by administering transfected Lin⁻HSC cells of thepresent invention by intravitreal injection of the cells into the eye.Such transfected Lin⁻HSC cells can comprise Lin⁻HSC transfected with atherapeutically useful gene, such as a gene encoding an antiangiogenicor neurotrophic gene product in addition to the TrpRS fragment.

Preferably, at least about 1×10⁵ transfected Lin⁻HSC cells areadministered by intravitreal injection to a mammalian eye suffering froma retinal degenerative disease. The number of cells to be injected maydepend upon the severity of the retinal degeneration, the age of themammal and other factors that will be readily apparent to one ofordinary skill in the art of treating retinal diseases. The transfectedLin⁻HSC may be administered in a single dose or by multiple doseadministration over a period of time, as determined by the clinician incharge of the treatment.

The Lin⁻HSCs of the present invention are useful for the treatment ofretinal injuries and retinal defects involving an interruption in ordegradation of the retinal vasculature or retinal neuronal degeneration.Human Lin⁻HSCs also can be used to generate a line of geneticallyidentical cells, i.e., clones, for use in regenerative or reparativetreatment of retinal vasculature, as well as for treatment oramelioration of retinal neuronal degeneration.

EXAMPLES Example 1 Cell Isolation and Enrichment; Preparation of MurineLin⁻HSC Populations A and B

General Procedure. All in vivo evaluations were performed in accordancewith the NIH Guide for the Care and Use of Laboratory Animals, and allevaluation procedures were approved by The Scripps Research Institute(TSRI, La Jolla, Calif.) Animal Care and Use Committee. Bone marrowcells were extracted from B6.129S7-Gtrosa26, Tie-2GFP, ACTbEGFP, FVB/NJ(rd/rd mice) or Balb/cBYJ adult mice (The Jackson Laboratory, ME).

Monocytes were then separated by density gradient separation usingHISTOPAQUE® polysucrose gradient (Sigma, St. Louis, Mo.) and labeledwith biotin conjugated lineage panel antibodies (CD45, CD3, Ly-6G, CD11,TER-119, Pharmingen, San Diego, Calif.) for Lin⁻ selection in mice.Lineage positive (Lin⁺) cells were separated and removed from Lin⁻HSCusing a magnetic separation device (AUTOMACS™ sorter, Miltenyi Biotech,Auburn, Calif.). The resulting Lin⁻HSC population, containingendothelial progenitor cells was further characterized using a FACS™Calibur flow cytometer (Becton Dickinson, Franklin Lakes, N.J.) usingfollowing antibodies: PE-conjugated-Sca-1, c-kit, KDR, and CD31(Pharmingen, San Diego, Calif.). Tie-2-GFP bone marrow cells were usedfor characterization of Tie-2.

To harvest adult mouse endothelial cells, mesenteric tissue wassurgically removed from ACTbEGFP mouse and placed in collagenase(Worthington, Lakewood, N.J.) to digest the tissue, followed byfiltration using a 45 μm filter. Flow-through was collected andincubated with Endothelial Growth Media (Clonetics, San Diego, Calif.).Endothelial characteristics were confirmed by observing morphologicalcobblestone appearance, staining with CD31 mAb (Pharmingen) andexamining cultures for the formation of tube-like structures inMATRIGEL™ matrix (Beckton Dickinson, Franklin Lakes, N.J.).

Murine Lin⁻HSC Population A. Bone marrow cells were extracted fromACTbEGFP mice by the General Procedure described above. The Lin⁻HSCcells were characterized by FACS flow cytometry for CD31, c-kit, Sca-1,Flk-1, and Tie-2 cell surface antigen markers. The results are shown inFIG. 1 (c). About 81% of the Lin⁻HSC exhibited the CD31 marker, about70.5% of the Lin HSC exhibited the c-kit marker, about 4% of the Lin⁻HSCexhibited the Sca-1 marker, about 2.2% of the Lin⁻HSC exhibited theFlk-1 marker and about 0.91% of the Lin⁻ HSC cell exhibited the Tie-2marker. In contrast, the Lin⁺HSC that were isolated from these bonemarrow cells had a significantly different cell marker profile (i.e.,CD31: 37.4%; c-kit: 20%; Sca-1: 2.8%; Flk-: 0.05%).

Murine Lin⁻HSC Population B. Bone marrow cells were extracted fromBalb/C, ACTbEGFP, and C3H mice by the General Procedure described above.The Lin⁻HSC cells were analyzed for the presence of cell surface markers(Sca-1, KDR, c-kit, CD34, CD31 and various integrins i.e., α₁, α₂, α₃,α₄, α₅, α₆, α_(M), α_(V), α_(X), α_(11b): β₁, β₄, β₃, β₄, β₅ and β₇).The results are shown in Table 1. TABLE 1 Characterization of Lin⁻ HSCPopulation B. Cell Marker Lin⁻ HSC integrin α₁ 0.10 integrin α₂ 17.57integrin α₃ 0.22 integrin α₄ 89.39 integrin α₅ 82.47 integrin α₆ 77.70integrin α_(L) 62.69 integrin α_(M) 35.84 integrin α_(X) 3.98 integrinα_(V) 33.64 integrin α_(IIb) 0.25 integrin β₁ 86.26 integrin β₂ 49.07integrin β₃ 45.70 integrin β₄ 0.68 integrin β₅ 9.44 integrin β₇ 11.25CD31 51.76 CD34 55.83 Flk-1/KDR 2.95 c-kit (CD117) 74.42 Sca-1 7.54

Example 2 Intravitreal Administration of Cells in a Murine Model

An eyelid fissure was created in a mouse eyelid with a fine blade toexpose the P2 to P6 eyeball. Lineage negative HSC Population A of thepresent invention (approximately 10⁵ cells in about 0.5 μl to about 1 μlof cell culture medium) was then injected intravitreally using a33-gauge (Hamilton, Reno, Nev.) needled-syringe.

Example 3 EPC Transfection

Murine Lin⁻HSC (Population A) were transfected with DNA encoding the T2fragment of TrpRS also enclosing a His₆ tag (SEQ ID NO: 1, FIG. 7) usingFuGENE™6 Transfection Reagent (Roche, Indianapolis, Ind.) according tomanufacturer's protocol. Lin⁻HSC cells (about 10⁶ cell per ml) weresuspended in opti-MEM® medium (Invitrogen, Carlsbad, Calif.) containingstem cell factor (PeproTech, Rocky Hill, N.J.). DNA (about 1 μg) andFuGENE reagent (about 3 μl) mixture was then added, and the mixtureswere incubated at about 37° C. for about 18 hours. After incubation,cells were washed and collected. The transfection rate of this systemwas approximately 17% that was confirmed by FACS analysis. T2 productionwas confirmed by western blotting. The amino acid sequence ofHis₆-tagged T2-TrpRS is shown as SEQ ID NO: 2, FIG. 8.

Example 4 Immunohistochemistry and Confocal Analysis

Mouse retinas were harvested at various time points and were preparedfor either whole mounting or frozen sectioning. For whole mounts,retinas were fixed with 4% paraformaldehyde, and blocked in 50% fetalbovine serum (FBS) and 20% normal goat serum for one hour at ambientroom temperature. Retinas were processed for primary antibodies anddetected with secondary antibodies. The primaries used were:anti-Collagen IV (Chemicon, Temecula, Calif., anti-β-gal (Promega,Madison, Wis.), anti-GFAP (Dako Cytomation, Carpenteria, Calif.),anti-α-smooth muscle actin (α-SMA, Dako Cytomation). Secondaryantibodies used were conjugated either to Alexa 488 or 594 fluorescentmarkers (Molecular Probes, Eugene, Oreg.). Images were taken using anMRC 1024 Confocal microscope (Bio-Rad, Hercules, Calif.).Three-dimensional images were created using LASERSHARP® software(Bio-Rad) to examine the three different layers of vascular developmentin the whole mount retina. The difference in GFP pixel intensity betweenenhanced GFP (eGFP) mice and GFAP/wtGFP mice, distinguished by confocalmicroscopy, was utilized to create the 3 dimensional images.

Example 5 In vivo Retinal Angiogenesis Quantification Assay in Mice

For T2-TrpRS analysis, the primary and deep plexus were reconstructedfrom the three dimensional images of mouse retinas. The primary plexuswas divided into two categories: normal development, or halted vascularprogression. The categories of inhibition of deep vascular developmentwere construed based upon the percentage of vascular inhibitionincluding the following criteria: complete inhibition of deep plexusformation was labeled “Complete”, normal vascular development (includingless than 25% inhibition) was labeled “Normal” and the remainder labeled“Partial.” For the rd/rd mouse rescue data, four separate areas of thedeeper plexus in each whole mounted retina were captured using a 10×lens. The total length of vasculature was calculated for each image,summarized and compared between the groups. To acquire accurateinformation, Lin⁻HSC were injected into one eye and Lin⁺HSC into anothereye of the same mouse. Non-injected control retinas were taken from thesame litter.

Example 6 Adult Retinal Injury Murine Models

Laser and scar models were created using either a diode laser (150 mW, 1second, 50 mm) or mechanically by puncturing the mouse retina with a 27gauge needle. Five days after injury, cells were injected using theintravitreal method. Eyes were harvested from the mice five days later.

Example 7 Neurotrophic Rescue of Retinal Regeneration

Adult, murine, bone marrow derived lineage negative hematopoietic stemcells (Lin⁻HSC) have a vasculotrophic and neurotrophic rescue effect ina mouse model of retinal degeneration. Right eyes of 10-day old micewere injected intravitreally with about 0.5 microliters containing about10⁵ Lin⁻HSC of the present invention and evaluated 2 months later forthe presence of retinal vasculature and neuronal layer nuclear count.The left eyes of the same mice were injected with about the same numberof Lin⁺HSC as a control, and were similarly evaluated. As shown in FIG.9, in the Lin⁻HSC treated eyes, the retinal vasculature appeared nearlynormal, the inner nuclear layer was nearly normal and the outer nuclearlayer (ONL) had about 3 to about 4 layers of nuclei. In contrast, thecontralateral Lin⁺HSC treated eye had a markedly atrophic middle retinalvascular layer, a completely atrophic outer retinal vascular layer; theinner nuclear layer was markedly atrophic and the outer nuclear layerwas completely gone. This was dramatically illustrated in Mouse 3 andMouse 5. In Mouse 1, there was no rescue effect and this was true forapproximately 15% of the injected mice.

When visual function was assessed with electroretinograms (ERG), therestoration of a positive ERG was observed when both the vascular andneuronal rescue was observed (Mice 3 and 5). Positive ERG was notobserved when there was no vascular or neuronal rescue (Mouse 1). Thiscorrelation between vascular and neurotrophic rescue of the rd/rd mouseeyes by the Lin⁻HSC of the present invention is illustrated by aregression analysis plot shown in FIG. 10. A correlation betweenneuronal (y-axis) and vascular (x-axis) recovery was observed for theintermediate vasculature type (r=0.45) and for the deep vasculature(r=0.67).

FIG. 11 shows the absence of any statistically significant correlationbetween vascular and neuronal rescue by Lin⁺HSC. The vascular rescue wasquantified and the data are presented in FIG. 12. Data for mice at 2month (1M), 2 months (2M), and 6 months (6M), post-injection shown inFIG. 12, demonstrate that vascular length was significantly increased ineyes treated with the Lin⁻HSC of the present invention (dark bars)relative to the vascular length in untreated eyes from the same mouse(light bars), particularly at 1 month and 2 months, post-injection. Theneurotrophic rescue effect was quantified by counting nuclei in theinner and outer nuclear layers about two months after injection ofLin⁻HSC or Lin⁺HSC. The results are presented in FIGS. 13 and 14.

Example 8 Human Lin⁻HSC Population

Bone marrow cells were extracted from healthy adult human volunteers bythe General Procedure described above. Monocytes were then separated bydensity gradient separation using HISTOPAQUE® polysucrose gradient(Sigma, St. Louis, Mo.). To isolate the Lin⁻HSC population from humanbone marrow mononuclear cells the following biotin conjugated lineagepanel antibodies were used with the magnetic separation system(AUTOMACS™ sorter, Miltenyi Biotech, Auburn, Calif.): CD2, CD3, CD4,CD11a, Mac-1, CD14, CD16, CD19, CD33, CD38, CD45RA, CD64, CD68, CD86,CD235a (Pharmingen).

The human Lin⁻HSC population was further separated into twosub-populations based on CD133 expression. The cells were labeled withbiotin-conjugated CD133 antibodies ans separated into CD133 positive andCD133 negative sub-populations.

Example 9 Intravitreal Administration of Human and Murine Cells inMurine Models for Retinal Degeneration

C3H/HeJ, C3SnSmn.CB17-Prkdc SCID, and rd10 mouse strains were used asretinal degeneration models. C3H/HeJ and C3SnSmn.CB17-Prkdc SCID mice(The Jackson Laboratory, Maine) were homozygous for the retinaldegeneration 1 (rd1) mutation, a mutation that causes early onset severeretinal degeneration. The mutation is located in exon 7 of the Pde6bgene encoding the rod photoreceptor cGMP phosphodiesterase β subunit.The mutation in this gene has been found in human patients withautosomal recessive retinitis pigmentosa (RP). C3SnSmn.CB17-Prkdc SCIDmice are also homozygous for the severe combined immune deficiencyspontaneous mutation (Prkdc SCID) and were used for human cell transferexperiments. Retinal degeneration in rd10 mice is caused by a mutationin exon 13 of Pde6b gene. This is also a clinically relevant RP modelwith later onset and milder retinal degeneration than rd1/rd1). Allevaluations were performed in accordance with the NIH Guide for the Careand Use of Laboratory Animals, and all procedures were approved by TheScripps Research Institute Animal Care and Use Committee.

An eyelid fissure was created in a mouse eyelid with a fine blade toexpose the P2 to P6 eyeball. Lineage negative HSC cells for murinepopulation A or human population C (approximately 10⁵ cells in about 0.5μl to about 1 μl of cell culture medium) were then injected in the mouseeye intravitreally using a 33-gauge (Hamilton, Reno, Nev.)needled-syringe. To visualize the injected human cells, cells werelabeled with dye (Cell tracker green CMFDA, Molecular Probes) beforeinjection.

Retinas were harvested at various time points and fixed with 4%paraformaldehyde (PFA) and methanol followed by blocking in 50% FBS/20%NGS for one hour at room temperature. To stain retinal vasculature,retinas were incubated with anti-CD31 (Pharmingen) and anti-collagen IV(Chemicon) antibodies followed by Alexa 488 or 594 conjugated secondaryantibodies (Molecular Probes, Eugene, Oreg.). The retinas were laid flatwith four radial relaxing incisions to obtain a whole mount preparation.Images of vasculature in intermediate or deep retinal vascular plexuses(see Dorrell et al. 2002 Invest Opthalmol. Vis. Sci. 43:3500-3510) wereobtained using a Radiance MP2100 confocal microscope and LASERSHARP®software (Biorad, Hercules, Calif.). For quantification of vasculature,four independent fields (900 μm×900 μm) were chosen randomly from themid-portion of the intermediate or deep vascular layer and the totallength of vasculature was measured using LASERPIX® analyzing software(Biorad). The total lengths of these four fields in the same plexus wereused for further analysis.

The flat-mounted retinas were re-embedded for cryostat sections. Retinaswere placed in 4% PFA overnight followed by incubation with 20% sucrose.The retinas were embedded in optimal cutting temperature compound (OCT:Tissue-Tek; Sakura FineTech, Torrance, Calif.). Cryostat sections (10μm) were re-hydrated in PBS containing the nuclear dye DAPI(Sigma-Aldrich, St. Louis, Mo.). DAPI-labeled nuclear images of threedifferent areas (280 μm width, unbiased sampling) in a single sectionthat contained optic nerve head and the entire peripheral retina weretaken by confocal microscope. The numbers of the nuclei located in ONLof the three independent fields in one section were counted and summedup for analysis. Simple linear-regression analysis was performed toexamine the relationship between the length of vasculature in the deepplexus and the number of cell nuclei in the ONL.

Following overnight dark-adaptation, mice were anesthetized byintraperitoneal injection of 15 μg/gm ketamine and 7 μg/gm xylazine.Electroretinograms (ERGs) were recorded from the corneal surface of eacheye after pupil dilation (1% atropine sulfate) using a gold loop cornealelectrode together with a mouth reference and tail ground electrode.Stimuli were produced with a Grass Photic Stimulator (PS33 Plus, GrassInstruments, Quincy, Mass.) affixed to the outside of a highlyreflective Ganzfeld dome. Rod responses were recorded toshort-wavelength (Wratten 47A; λ_(max)=470 nm) flashes of light over arange of intensities up to the maximum allowable by the photicstimulator (0.668 cd-s/m²). Response signals were amplified (CP511 ACamplifier, Grass Instruments), digitized (PCI-1200, NationalInstruments, Austin, Tex.) and computer-analyzed. Each mouse served asits own internal control with ERGs recorded from both the treated anduntreated eyes. Up to 100 sweeps were averaged for the weakest signals.The averaged responses from the untreated eye were digitally subtractedfrom the responses from the treated eye and this difference in signalwas used to index functional rescue.

Microarray analysis was used for evaluation of Lin⁻HSC-targeted retinalgene expression. P6 rd/rd mice were injected with either Lin⁻ or CD31⁻HSCs. The retinas of these mice were dissected 40 days post-injection inRNase free medium (rescue of the retinal vasculature and thephotoreceptor layer is obvious at this time point after injection). Onequadrant from each retina was analyzed by whole mount to ensure thatnormal HSC targeting as well as vasculature and neural protection hadbeen achieved. RNA from retinas with successful injections was purifiedusing a TRIZOL® phenol/chloroform RNA isolation protocol (LifeTechnologies, Rockville, Md.). RNA was hybridized to Affymetrix Mu74Av2chips and gene expression was analyzed using GENESPRING® software(SiliconGenetics, Redwood City, Calif.). Purified human or mouse HSCswere injected intravitreally into P6 mice. At P45 the retinas weredissected and pooled into fractions of 1) human HSC-injected, rescuedmouse retinas, 2) human HSC-injected, non-rescued mouse retinas, and 3)mouse HSC-injected, rescued mouse retinas for purification of RNA andhybridization to human-specific U133A Affymetrix chips. GENESPRING®software was used to identify genes that were expressed above backgroundand with higher expression in the human HSC-rescued retinas. Theprobe-pair expression profiles for each of these genes were thenindividually analyzed and compared to a model of normal human U133Amicroarray experiments using dChip to determine human species specifichybridization and to eliminate false positives due to cross-specieshybridization.

FIG. 21 illustrates flow cytometry data comparing the expression of CD31and integrin α₆ surface antigens on CD133 positive (DC133⁺) and CD133negative (CD133⁻) human Lin⁻ HSC populations of the present invention.The left panels show flow cytometry scatter plots. The center and rightpanels are histograms showing the level of specific antibody expressionon the cell population. The Y axis represents the number of events andthe X axis shows the intensity of the signal. The outlined histogramsare isotype IgG control antibodies showing the level of non-specificbackground staining. The filled histograms show the level of specificantibody expression on the cell population. A filled histogram shiftedto the right of the outlined (control) histogram represents an increasedfluorescent signal and expression of the antibody above backgroundlevel. Comparing the position of the peaks of the filled histogramsbetween the two cell populations represents the difference in proteinexpression on the cells. For example, CD31 is expressed above backgroundon both CD133⁺ and CD133⁻ cells of the invention; however, there aremore cells expressing lower levels of CD31 in the CD133⁺ cell populationthan there are in the CD133⁻ population. From this data it is evidentthat CD31 expression varies between the two populations and that thealpha 6 integrin expression is largely limited to cells in theLin-population, and thus may serve as a marker of cells with vasculo-and neurotrophic rescue function.

When the CD133 positive and CD133 negative Lin⁻ HSC sub-population wasintravitreally injected into the eyes of neonatal SCID mice, thegreatest extent of incorporation into the developing vasculature wasobserved for the CD133 negative sub-population, which expresses bothCD31 and integrin α₆ surface antigens (see FIG. 21, bottom). The CD133positive sub-population, which does not express CD31 or integrin α₆(FIG. 21, top) appears to target sites of peripheral ischemia-drivenneovascularization, but not when injected into eyes undergoingangiogenesis.

Rescued and non-rescued retinas were analyzed immunohistochemically withantibodies specific for rod or cone opsin. The same eyes used for theERG recordings presented in FIG. 17 were analyzed for rod or cone opsin.In wild type mouse retinas, less than 5% of photoreceptors present arecones (Soucy et al. 1998, Neuron 21: 481-493) and theimmunohistochemical staining patterns observed with red/green cone opsinas shown in FIG. 25, panel (A) or rod rhodopsin as shown in FIG. 25,panel (B), were consistent with this percentage of cone cells.Antibodies specific for rod rhodopsin (rho4D2) were provided by Dr.Robert Molday of the University of British Columbia and used asdescribed previously (Hicks et al. 1986, Exp. Eye Res. 42: 55-71).Rabbit antibodies specific for cone red/green opsin were purchased fromChemicon (AB5405) and used according to the manufacturer's instructions.

Example 10 Intravitreal Administration of Murine Cells in Murine Modelsfor Oxygen Induced Retinal Degeneration

New born wild-type C57B16 mice were exposed to hyperoxia (75% oxygen)between postnatal days P7 to P12 in an oxygen-induced retinaldegeneration (OIR) model. FIG. 22 illustrates normal postnatal vasculardevelopment in C57B16 mice from P0 to P30. At P0 only buddingsuperficial vessels can be observed around the optic disc. Over the nextfew days, the primary superficial network extends toward the periphery,reaching the far periphery by day P10. Between P7 and P12, the secondary(deep) plexus develops. By P17, an extensive superficial and deepnetwork of vessels is present (FIG. 22, insets). In the ensuing days,remodeling occurs along with development of the tertiary (intermediate)layer of vessels until the adult structure is reached approximately atP21.

In contrast, in the OIR model, following exposure to 75% oxygen atP7-P12, the normal sequence of events is severely disrupted (FIG. 23).Adult murine Lin⁻HSC populations of the invention were intravitreallyinjected at P3 in an eye of a mouse that was subsequently subjected toOIR, the other eye was injected with PBS or CD31 negative cells as acontrol. FIG. 24 illustrates that the Lin⁻HSC populations of the presentinvention can reverse the degenerative effects of high oxygen levels inthe developing mouse retina. Fully developed superficial and deepretinal vasculature was observed at P17 in the treated eyes, whereas inthe control eyes showed large avascular areas with virtually no deepvessels (FIG. 24). Approximately 100 eyes of mice in the OIR model wereobserved. Normal vascularization was observed in 58% of the eyes treatedwith the Lin⁻HSC populations of the invention, compared to 12% of thecontrol eyes treated with CD31⁻ cells and 3% of the control eyes treatedwith PBS.

Results and Discussion

Murine Retinal Vascular Development; A Model for Ocular Angiogenesis.The mouse eye provides a recognized model for the study of mammalianretinal vascular development, such as human retinal vasculardevelopment. During development of the murine retinal vasculature,ischemia-driven retinal blood vessels develop in close association withastrocytes. These glial elements migrate onto the third trimester humanfetus, or the neonatal rodent, retina from the optic disc along theganglion cell layer and spread radially. As the murine retinalvasculature develops, endothelial cells utilize this already establishedastrocytic template to determine the retinal vascular pattern (See FIG.1, panel (a and b)). FIG. 1 depicts schematic diagrams of developingmouse retina. Panel (a) depicts development of the primary plexus (darklines at upper left of the diagram) superimposed over the astrocytetemplate (light lines) whereas, panel (b) depicts the second phase ofretinal vessel formation. In FIG. 1, GCL stands for ganglion cell layer;IPL stands for inner plexus layer; INL stands for inner nuclear layer;OPL stands for outer plexus layer; ONL stands for outer nuclear layer;RPE stands for retinal pigment epithelium; ON stands for optic nerve;and P stands for periphery.

At birth, retinal vasculature is virtually absent. By postnatal day 14(P14) the retina has developed complex primary (superficial) andsecondary (deep) layers of retinal vessels coincident with the onset ofvision. Initially, spoke-like peripapillary vessels grow radially overthe pre-existing astrocytic network towards the periphery, becomingprogressively interconnected by capillary plexus formation. Thesevessels grow as a monolayer within the nerve fiber through P10 (FIG. 1,panel (a)). Between P7-P8 collateral branches begin to sprout from thisprimary plexus and penetrate into the retina to the outer plexiformlayer where they form the secondary, or deep, retinal plexus. By P21,the entire network undergoes extensive remodeling and a tertiary, orintermediate, plexus forms at the inner surface of inner nuclear layer(FIG. 1, panel (b)).

The neonatal mouse retinal angiogenesis model is useful for studying therole of HSC during ocular angiogenesis for several reasons. In thisphysiologically relevant model, a large astrocytic template exists priorto the appearance of endogenous blood vessels, permitting an evaluationof the role for cell-cell targeting during a neovascular process. Inaddition, this consistent and reproducible neonatal retinal vascularprocess is known to be hypoxia-driven, in this respect havingsimilarities to many retinal diseases in which ischemia is known to playa role.

Enrichment of Endothelial Progenitor Cells (EPC) from Bone Marrow.Although cell surface marker expression has been extensively evaluatedon the EPC population found in preparations of HSC, markers thatuniquely identify EPC are still poorly defined. To enrich for EPC,hematopoietic lineage marker positive cells (Lin⁺), i.e., B lymphocytes(CD45), T lymphocytes (CD3), granulocytes (Ly-6G), monocytes (CD11), anderythrocytes (TER-119), were depleted from bone marrow mononuclear cellsof mice. Sca-1 antigen was used to further enrich for EPC. A comparisonof results obtained after intravitreal injection of identical numbers ofeither Lin⁻Sca-1⁺ cells or Lin cells, no difference was detected betweenthe two groups. In fact, when only Lin⁻Sca-1⁻ cells were injected, fargreater incorporation into developing blood vessels was observed.

The Lin⁻HSC populations of the present invention are enriched with EPCs,based on functional assays. Furthermore, Lin⁺HSC populationsfunctionally behave quite differently from the Lin HSC populations.Epitopes commonly used to identify EPC for each fraction (based onpreviously reported in vitro characterization studies) were alsoevaluated. While none of these markers were exclusively associated withthe Lin⁻ fraction, all were increased about 70 to about 1800% in theLin⁻HSC, compared to the Lin⁺HSC fraction. FIG. 1, panel (c) illustratesflow cytometric characterization of bone marrow-derived Lin⁺HSC andLin⁻HSC separated cells. The top row of panel (c) shows a hematopoieticstem cell dot plot distribution of non-antibody labeled cells. R1defines the quantifiable-gated area of positive PE-staining; R2indicates GFP-positive. Dot plots of Lin⁻HSC are shown in the middle rowand dot plots of Lin⁺HSC are shown in the bottom row. The C57B/6 cellswere labeled with the PE-conjugated antibodies for Sca-1, c-kit,Flk-1/KDR, CD31. Tie-2 data was obtained from Tie-2-GFP mice. Thepercentages in the corners of the dot plots indicate the percent ofpositive-labeled cells out of total Lin or Lin⁺HSC population.Interestingly, accepted EPC markers like Flk-1/KDR, Tie-2, and Sca-1were poorly expressed and, thus, were not used for furtherfractionation.

Intravitreally Injected HSC Lin⁻Cells Contain EPC That Target Astrocytesand Incorporate into Developing Retinal Vasculature. To determinewhether intravitreally injected Lin⁻HSC can target specific cell typesof the retina, utilize the astrocytic template and participate inretinal angiogenesis, approximately 10⁵ cells from a Lin⁻HSC compositionof the present invention or Lin⁺HSC cells (control, about 10⁵ cells)isolated from the bone marrow of adult (GFP or LacZ transgenic) micewere injected into postnatal day 2 (P2) mouse eyes. Four days afterinjection (P6), many cells from the Lin⁻HSC composition of the presentinvention, derived from GFP or LacZ transgenic mice were adherent to theretina and had the characteristic elongated appearance of endothelialcells. FIG. 2 illustrates engraftment of Lin⁻ cells into developingmouse retina. As shown in FIG. 2, panel (a), the four dayspost-injection (P6) intravitreally injected eGFP+Lin⁻HSC attach anddifferentiate on the retina.

In many areas of the retinas, the GFP-expressing cells were arranged ina pattern conforming to underlying astrocytes and resembled bloodvessels. These fluorescent cells were observed ahead of the endogenous,developing vascular network (FIG. 2, panel (b)). Conversely, only asmall number of Lin⁺HSC (FIG. 2, panel (c)), or adult mouse mesentericendothelial cells (FIG. 2, panel (d)) attached to the retinal surface.In order to determine whether cells from an injected Lin⁻HSC populationcould also attach to retinas with already established vessels, weinjected a Lin⁻HSC composition into adult eyes. Interestingly, no cellswere observed to attach to the retina or incorporate into established,normal retinal blood vessels (FIG. 2, panel (e)). This indicates thatthe Lin⁻HSC compositions of the present invention do not disrupt anormally developed vasculature and will not initiate abnormalvascularization in normally developed retinas.

In order to determine the relationship between an injected Lin⁻HSCcompositions of the present invention and retinal astrocytes, atransgenic mouse was used, which expressed glial fibrillary acidicprotein (GFAP, a marker of astrocytes) and promoter-driven greenfluorescent protein (GFP). Examination of retinas of these GFAP-GFPtransgenic mice injected with Lin⁻HSC from eGFP transgenic micedemonstrated co-localization of the injected eGFP EPC and existingastrocytes (FIG. 2, panel (f-h), arrows). Processes of eGFP⁺Lin⁻HSC wereobserved to conform to the underlying astrocytic network (arrows, FIG.2, panel (g)). Examination of these eyes demonstrated that the injected,labeled cells only attached to astrocytes; in P6 mouse retinas, wherethe retinal periphery does not yet have endogenous vessels, injectedcells were observed adherent to astrocytes in these not yet vascularizedareas. Surprisingly, injected, labeled cells were observed in the deeperlayers of the retina at the precise location where normal retinalvessels will subsequently develop (FIG. 2, panel (i), arrows).

To determine whether injected Lin⁻HSC of the present invention arestably incorporated into the developing retinal vasculature, retinalvessels at several later time points were examined. As early as P9(seven days after injection), Lin⁻HSC incorporated into CD31⁺ structures(FIG. 2, panel (j)). By P16 (14 days after injection), the cells werealready extensively incorporated into retinal vascular-like structures(FIG. 2, panel (k)). When rhodamine-dextran was injected intravascularly(to identify functional retinal blood vessels) prior to sacrificing theanimals, the majority of Lin⁻HSC were aligned with patent vessels (FIG.2, panel (1)). Two patterns of labeled cell distribution were observed:(1) in one pattern, cells were interspersed along vessels in betweenunlabeled endothelial cells; and (2) the other pattern showed thatvessels were composed entirely of labeled cells. Injected cells werealso incorporated into vessels of the deep vascular plexus (FIG. 2,panel (m)). While sporadic incorporation of Lin⁻HSC-derived EPC intoneovasculature has been previously reported, this is the first report ofvascular networks being entirely composed of these cells. Thisdemonstrates that cells from a population of bone marrow-derived Lin⁻HSCof the present invention injected intravitreally can efficientlyincorporate into any layer of the forming retinal vascular plexus.

Histological examination of non-retinal tissues (e.g., brain, liver,heart, lung, bone marrow) did not demonstrate the presence of any GFPpositive cells when examined up to 5 or 10 days after intravitrealinjection. This indicates that a sub-population of cells within theLin⁻HSC fraction selectively target to retinal astrocytes and stablyincorporate into developing retinal vasculature. Since these cells havemany characteristics of endothelial cells (association with retinalastrocytes, elongate morphology, stable incorporation into patentvessels and not present in extravascular locations), these cellsrepresent EPC present in the Lin⁻HSC population. The targeted astrocytesare of the same type observed in many of the hypoxic retinopathies. Itis well known that glial cells are a prominent component of neovascularfronds observed in DR and other forms of retinal injury. Underconditions of reactive gliosis and ischemia-induced neovascularization,activated astrocytes proliferate, produce cytokines, and up-regulateGFAP, similar to that observed during neonatal retinal vascular templateformation in many mammalian species including humans.

Lin⁻HSC populations of the present invention will target activatedastrocytes in adult mouse eyes as they do in neonatal eyes, Lin⁻HSCcells were injected into adult eyes with retinas injured byphoto-coagulation (FIG. 3, panel (a)) or needle tip (FIG. 3, panel (b)).In both models, a population of cells with prominent GFAP staining wasobserved only around the injury site (FIG. 3, panels (a and b)). Cellsfrom injected Lin⁻HSC compositions localized to the injury site andremained specifically associated with GFAP-positive astrocytes (FIG. 3,panels (a and b)). At these sites, Lin⁻HSC cells were also observed tomigrate into the deeper layer of retina at a level similar to thatobserved during neonatal formation of the deep retinal vasculature.Uninjured portions of retina contained no Lin⁻HSC cells, identical tothat observed when Lin⁻HSC were injected into normal, uninjured adultretinas (FIG. 2, panel (e)). These data indicate that Lin⁻HSCcompositions can selectively target activated glial cells in injuredadult retinas with gliosis as well as neonatal retinas undergoingvascularization.

Intravitreally Injected Lin⁻HSC Can Rescue and Stabilize DegeneratingVasculature. Since intravitreally injected Lin⁻HSC compositions targetastrocytes and incorporate into the normal retinal vasculature, thesecells also stabilize degenerating vasculature in ischemic ordegenerative retinal diseases associated with gliosis and vasculardegeneration. The rd/rd mouse is a model for retinal degeneration thatexhibits profound degeneration of photoreceptor and retinal vascularlayers by one month after birth. The retinal vasculature in these micedevelops normally until P16 at which time the deeper vascular plexusregresses; in most mice the deep and intermediate plexuses have nearlycompletely degenerated by P30.

To determine whether HSC can rescue the regressing vessels, Lin⁺ orLin⁻HSC (from Balb/c mice) were injected into rd/rd mice intravitreallyat P6. By P33, after injection with Lin⁺ cells, vessels of the deepestretinal layer were nearly completely absent (FIG. 4, panel (a and b)).In contrast, most Lin⁻HSC-injected retinas by P33 had a nearly normalretinal vasculature with three parallel, well-formed vascular layers(FIG. 4, panel (a and d)). Quantification of this effect demonstratedthat the average length of vessels in the deep vascular plexus of Lin⁻injected rd/rd eyes was nearly three times greater than untreated orLin⁺ cell-treated eyes (FIG. 4, panel (e)). Surprisingly, injection of aLin⁻HSC composition derived from rd/rd adult mouse (FVB/N) bone marrowalso rescued degenerating rd/rd neonatal mouse retinal vasculature (FIG.4, panel (f)). Degeneration of the vasculature in rd/rd mouse eyes inobserved as early as 2-3 weeks post-naturally. Injection of Lin⁻ HSC aslate as P15 also resulted in partial stabilization of the degeneratingvasculature in the rd/rd mice for at least one month (FIG. 4, panels (gand h)).

A Lin⁻HSC composition injected into younger (e.g., P2) rd/rd mice alsoincorporated into the developing superficial vasculature. By P11, thesecells were observed to migrate to the level of the deep vascular plexusand form a pattern identical to that observed in the wild type outerretinal vascular layer (FIG. 5, panel (a)). In order to more clearlydescribe the manner in which cells from injected Lin⁻HSC compositionsincorporate into, and stabilize, degenerating retinal vasculature in therd/rd mice, a Lin⁻HSC composition derived from Balb/c mice was injectedinto Tie-2-GFP FVB mouse eyes. The FVB mice have the rd/rd genotype andbecause they express the fusion protein Tie-2-GFP, all endogenous bloodvessels are fluorescent.

When non-labeled cells from a Lin⁻HSC composition are injected intoneonatal Tie-2-GFP FVB eyes and are subsequently incorporated into thedeveloping vasculature, there should be non-labeled gaps in theendogenous, Tie-2-GFP labeled vessels that correspond to theincorporated, non-labeled Lin⁻HSC that were injected. Subsequentstaining with another vascular marker (e.g., CD-31) then delineates theentire vessel, permitting determination as to whether non-endogenousendothelial cells are part of the vasculature. Two months afterinjection, CD31-positive, Tie-2-GFP negative, vessels were observed inthe retinas of eyes injected with the Lin⁻HSC composition (FIG. 5, panel(b)). Interestingly, the majority of rescued vessels contained Tie-2-GFPpositive cells (FIG. 5, panel (c)). The distribution of pericytes, asdetermined by staining for smooth muscle actin, was not changed byLin⁻HSC injection, regardless of whether there was vascular rescue (FIG.5, panel (d)). These data clearly demonstrate that intravitreallyinjected Lin⁻HSC compositions of the present invention migrate into theretina, participate in the formation of normal retinal blood vessels,and stabilize endogenous degenerating vasculature in a geneticallydefective mouse.

Inhibition of Retinal Angiogenesis by Transfected Cells from Lin⁻HSC.The majority of retinal vascular diseases involve abnormal vascularproliferation rather than degeneration. Transgenic cells targeted toastrocytes can be used to deliver an anti-angiogenic protein and inhibitangiogenesis. Cells from Lin⁻HSC compositions were transfected withT2-tryptophanyl-tRNA synthetase (T2-TrpRS). T2-TrpRS is a 43 kD fragmentof TrpRS that potently inhibits retinal angiogenesis (FIG. 6, panel(a)). On P12, retinas of eyes injected with a controlplasmid-transfected Lin⁻HSC composition (no T2-TrpRS gene) on P2 hadnormal primary (FIG. 6, panel (c)) and secondary (FIG. 6, panel (d))retinal vascular plexuses. When the T2-TrpRS transfected Lin⁻HSCcomposition of the present invention was injected into P2 eyes andevaluated 10 days later, the primary network had significantabnormalities (FIG. 6, panel (e)) and formation of the deep retinalvasculature was nearly completely inhibited (FIG. 6, panel (f)). The fewvessels observed in these eyes were markedly attenuated with large gapsbetween vessels. The extent of inhibition by T2-TrpRS-secreting Lin⁻HSCsis detailed in Table 2.

T2-TrpRS is produced and secreted by cells in the Lin⁻HSC composition invitro and after injection of these transfected cells into the vitreous,a 30 kD fragment of T2-TrpRS in the retina (FIG. 6, panel (b)) wasobserved. This 30 kD fragment was specifically observed only in retinasinjected with transfected Lin⁻HSC of the present invention and thisdecrease in apparent molecular weight compared to the recombinant or invitro-synthesized protein may be due to processing or degradation of theT2-TrpRS in vivo. These data indicate that Lin⁻HSC compositions can beused to deliver functionally active genes, such as genes expressingangiostatic molecules, to the retinal vasculature by targeting toactivated astrocytes. While it is possible that the observed angiostaticeffect is due to cell-mediated activity this is very unlikely since eyestreated with identical, but non-T2-transfected Lin⁻HSC compositions hadnormal retinal vasculature. TABLE 2 Vascular Inhibition byT2-TrpRS-secreting Lin⁻HSCs Primary Plexus Deep Plexus Inhibited NormalComplete Partial Normal T2TrpRS 60%  40% 33.3%   60%  6.7% (15 eyes) (9eyes)  (6 eyes) (5 eyes) (9 eyes) (1 eye)  Control  0% 100%   0% 38.5%61.5% (13 eyes) (0 eyes) (13 eyes) (0 eyes) (5 eyes) (8 eyes)

Intravitreally injected Lin⁻HSC populations localize to retinalastrocytes, incorporate into vessels, and can be useful in treating manyretinal diseases. While most cells from injected HSC compositions adhereto the astrocytic template, small numbers migrate deep into the retina,homing to regions where the deep vascular network will subsequentlydevelop. Even though no GFAP-positive astrocytes were observed in thisarea prior to 42 days postnatally, this does not rule out thepossibility that GFAP-negative glial cells are already present toprovide a signal for Lin⁻HSC localization. Previous studies have shownthat many diseases are associated with reactive gliosis. In DR, inparticular, glial cells and their extracellular matrix are associatedwith pathological angiogenesis.

Since cells from injected Lin⁻HSC compositions specifically attached toGFAP-expressing glial cells, regardless of the type of injury, Lin⁻HSCcompositions of the present invention can be used to targetpre-angiogenic lesions in the retina. For example, in the ischemicretinopathies such as diabetes, neovascularization is a response tohypoxia. By targeting Lin⁻HSC compositions to sites of pathologicalneovascularization, developing neovasculature can be stabilizedpreventing abnormalities of neovasculature such as hemorrhage or edema(the causes of vision loss associated with DR) and can potentiallyalleviate the hypoxia that originally stimulated the neovascularization.Abnormal blood vessels can be restored to normal condition. Furthermore,angiostatic proteins, such as T2-TrpRS can be delivered to sites ofpathological angiogenesis by using transfected Lin⁻HSC compositions andlaser-induced activation of astrocytes. Since laser photocoagulation isa commonly used in clinical opthalmology, this approach has applicationfor many retinal diseases. While such cell-based approaches have beenexplored in cancer therapy, their use for eye diseases is moreadvantageous since intraocular injection makes it possible to deliverlarge numbers of cells directly to the site of disease.

Neurotrophic and Vasculotrophic Rescue by Lin⁻HSC. MACS was used toseparate Lin⁻HSC from bone marrow of enhanced green fluorescent protein(eGFP), C3H (rd/rd), FVB (rd/rd) mice as described above. Lin⁻HSCcontaining EPC from these mice were injected intravitreally into P6 C3Hor FVB mouse eyes. The retinas were collected at various time points (1month, 2 months, and 6 months) after injection. The vasculature wasanalyzed by scanning laser confocal microscope after staining withantibodies to CD31 and retinal histology after nuclear staining withDAPI. Microarray gene expression analysis of mRNA from retinas atvarying time points was also used to identify genes potentially involvedin the effect.

Eyes of rd/rd mice had profound degeneration of both neurosensory retinaand retinal vasculature by P21. Eyes of rd/rd mice treated with Lin⁻HSCon P6 maintained a normal retinal vasculature for as long as 6 months;both deep and intermediate layers were significantly improved whencompared to the controls at all time points (1M, 2M, and 6M) (see FIG.12). In addition, we observed that retinas treated with Lin⁻HSC werealso thicker (1M; 1.2-fold, 2M; 1.3-fold, 6M; 1.4-fold) and had greaternumbers of cells in the outer nuclear layer (1M; 2.2-fold, 2M; 3.7-fold,6M; 5.7-fold) relative to eyes treated with Lin⁺HSC as a control. Largescale genomic analysis of “rescued” (e.g., Lin⁻HSC) compared to control(untreated or non-Lin⁻ treated) rd/rd retinas demonstrated a significantupregulation of genes encoding sHSPs (small heat shock proteins) andspecific growth factors that correlated with vascular and neural rescue,including genes encoding the proteins listed in FIG. 20, panels A and B.

The bone marrow derived Lin⁻HSC populations of the present inventionsignificantly and reproducibly induced maintenance of a normalvasculature and dramatically increased photoreceptor and other neuronalcell layers in the rd/rd mouse. This neurotrophic rescue effectcorrelated with significant upregulation of small heat shock proteinsand growth factors and provides insights into therapeutic approaches tocurrently untreatable retinal degenerative disorders.

Rd1/rd1 Mouse Retinas Exhibit Profound Vascular and NeuronalDegeneration. Normal postnatal retinal vascular and neuronal developmentin mice has been well described and is analogous to changes observed inthe third trimester human fetus (Dorrell et al., 2002, Invest.Opthalmol. Vis. Sci. 43:3500-3510). Mice homozygous for the rd1 geneshare many characteristics of human retinal degeneration (Frasson etal., 1999, Nat. Med. 5:1183-1187) and exhibit rapid photoreceptor (PR)loss accompanied by severe vascular atrophy as the result of a mutationin the gene encoding PR cGMP phosphodiesterase (Bowes et al. 1990,Nature 347:677-680). To examine the vasculature during retinaldevelopment and its subsequent degeneration, antibodies against collagenIV (CIV), an extracellular matrix (ECM) protein of mature vasculature,and CD31 (PECAM-1), a marker for endothelial cells were used (FIG. 15).Retinas of rd1/rd1 (C3H/HeJ) developed normally until approximatelypostnatal day (P) 8 when degeneration of the photoreceptor-containingouter nuclear layer (ONL) began. The ONL rapidly degenerated and cellsdied by apoptosis such that only a single layer of nuclei remained byP20. Double staining of the whole-mounted retinas with antibodies toboth CIV and CD31 revealed details of the vascular degeneration inrd1/rd1 mice similar to that described by others (Blanks et al., 1986,J. Comp. Neurol. 254:543-553). The primary and deep retinal vascularlayers appeared to develop normally though P12 after which there is arapid loss of endothelial cells as evidenced by the absence of CD31staining. CD31 positive endothelial cells were present in a normaldistribution through P12 but rapidly disappeared after that.Interestingly, CIV positive staining remained present throughout thetime points examined, suggesting that the vessels and associated ECMformed normally, but only the matrix remained after P13 by which time noCD31 positive cells were observed. (FIG. 15, middle panels). Theintermediate vascular plexus also degenerates after P21, but theprogression is slower than that observed in the deep plexus (FIG. 15,upper panel). Retinal vascular and neural cell layers of a normal mouseare shown for comparison to the rd1/rd1 mouse (right panels, FIG. 15).

Neuroprotective Effect of Bone Marrow-Derived Lin⁻HSCs in rd1/rd1 Mice.Intravitreally injected Lin⁻HSCs incorporate into endogenous retinalvasculature in all three vascular plexuses and prevent the vessels fromdegenerating. Interestingly, the injected cells are virtually neverobserved in the outer nuclear layer. These cells either incorporate intothe forming retinal vessels or are observed in close proximity to thesevessels. Murine Lin HSCs (from C3H/HeJ) were intravitreally injectedinto C3H/HeJ (rd1/rd1) mouse eyes at P6, just prior to the onset ofdegeneration. By P30, control cell (CD31⁻)-injected eyes exhibited thetypical rd1/rd1 phenotype, i.e., nearly complete degeneration of thedeep vascular plexus and ONL was observed in every retina examined. Eyesinjected with Lin HSCs maintained normal-appearing intermediate and deepvascular plexuses. Surprisingly, significantly more cells were observedin the internuclear layer (INL) and ONL of Lin⁻ HSC-injected eyes thanin control cell-injected eyes (FIG. 16, panel (A)). This rescue effectof Lin⁻HSCs could be observed at 2 months (FIG. 16, panel (B)) and foras long as 6 months after injection (FIG. 16, panel (C)). Differences inthe vasculature of the intermediate and deep plexuses ofLin⁻HSC-injected eyes, as well as the neuronal cell-containing INL andONL, were significant at all time points measured when rescued andnon-rescued eyes were compared (FIG. 16, panels (B and C)). This effectwas quantified by measuring the total length of the vasculature (FIG.16, panel (D)) and counting the number of DAPI-positive cell nucleiobserved in the ONL (FIG. 16, panel (E)). Simple linear-regressionanalysis was applied to the data at all time points.

A statistically significant correlation was observed between vascularrescue and neuronal (e.g., ONL thickness) rescue at P30 (p<0.024) andP60 (p<0.034) in the Lin³¹ HSC-injected eyes (FIG. 16 (F)). Thecorrelation remained high, although not statistically significant(p<0.14) at P180 when comparing Lin⁻HSC-injected retinas to controlcell-injected retinas (FIG. 16, panel (F)). In contrast, controlcell-injected retinas showed no significant correlation between thepreservation of vasculature and ONL at any time point (FIG. 16, panel(F)). These data demonstrate that intravitreal injection of Lin⁻HSCsresults in concomitant retinal vascular and neuronal rescue in retinasof rd1/rd1 mice. Injected cells were not observed in the ONL or anyplace other than within, or in close proximity to, retinal bloodvessels.

Functional Rescue of Lin⁻HSC-injected rd/rd Retinas. Electroretinograms(ERGs) were performed on mice 2 months after injection of control cellsor murine Lin HSCs (FIG. 17). Immunohistochemical and microscopicanalysis was done with each eye following ERG recordings to confirm thatvascular and neuronal rescue had occurred. Representative ERG recordingsfrom treated, rescued and control, non-rescued eyes show that in therescued eyes, the digitally subtracted signal (treated minus untreatedeyes) produced a clearly detectable signal with an amplitude on theorder of 8-10 microvolts (FIG. 17). Clearly, the signals from both eyesare severely abnormal. However, consistent and detectable ERGs wererecordable from the Lin⁻HSC-treated eyes. In all cases the ERG from thecontrol eye was non-detectable. While the amplitudes of the signals inrescued eyes were considerably lower than normal, the signals wereconsistently observed whenever there was histological rescue and were onthe order of magnitude of those reported by other, gene based, rescuestudies. Overall these results are demonstrate of some degree offunctional rescue in the eyes treated with the Lin⁻HSCs of theinvention.

Rescued rd/rd retinal cell types are predominantly cones. Rescued andnon-rescued retinas were analyzed immunohistochemically with antibodiesspecific for rod or cone opsin. The same eyes used for the ERGrecordings presented in FIG. 17 were analyzed for rod or cone opsin. Inwild type mouse retinas, less than about 5% of photoreceptors presentare cones (Soucy et al. 1998, Neuron 21: 481-493) and theimmunohistochemical staining patterns observed with red/green cone opsinas shown in FIG. 25, panel (A) or rod rhodopsin as shown in FIG. 25,panel (B), were consistent with this percentage of cone cells. When wildtype retinas were stained with pre-immune IgG, no staining was observedanywhere in the neurosensory retinas other than autoflouresence of theblood vessels (FIG. 25, panel (C)). Two months after birth, retinas ofnon-injected rd/rd mice had an essentially atrophic outer nuclear layerthat does not exhibit any staining with antibodies to red green coneopsin (FIG. 25, panel (D)) or rhodopsin (FIG. 25, panel (G)). Eyesinjected with control, CD31-HSC also did not stain positively for thepresence of either cone (FIG. 25, panel (E))) or rod (FIG. 25, panel(H)) opsin. In contrast, contralateral eyes injected with Lin-HSC hadabout 3 to about 8 rows of nuclei in a preserved outer nuclear layer;most of these cells were positive for cone opsin (FIG. 25, panel (F))with approximately 1-3% positive for rod opsin (FIG. 25, panel (I)).Remarkably, this is nearly the reverse of what is ordinarily observed inthe normal mouse retina, which is rod-dominated. These data demonstratethat the injection of Lin-HSC preserves cones for extended periods oftime during which they would ordinarily degenerate.

Human bone marrow (hBM)-derived Lin⁻HSCs also Rescue DegeneratingRetinas. Lin⁻ HSCs isolated from human bone marrow behave similarly tomurine Lin⁻HSCs. Bone marrow was collected from human donors and theLin⁺HSCs were depleted, producing a population of human Lin⁻HSCs(hLin⁻HSCs). These cells were labeled ex-vivo with fluorescent dye andinjected into C3SnSmn.CB17-Prkdc SCID mouse eyes. The injected hLin⁻HSCsmigrated to, and targeted, sites of retinal angiogenesis in a fashionidentical to that observed when murine Lin⁻HSCs were injected (FIG. 18,panel (A)). In addition to the vascular targeting, the human Lin⁻HSCsalso provided a robust rescue effect on both the vascular and neuronalcell layers of the rd1/rd1 mice (FIG. 18, panel (B and C)). Thisobservation confirms the presence of cells in human bone marrow thattarget retinal vasculature and can prevent retinal degeneration.

Lin⁻HSCs have Vasculo- and Neurotrophic Effects in the rd10/rd10 Mouse.While the rd1/rd1 mouse is the most widely used and best characterizedmodel for retinal degeneration (Chang et al. 2002, Vision Res.42:517-525), the degeneration is very rapid and in this regard differsfrom the usual, slower time course observed in the human disease. Inthis strain, photoreceptor cell degeneration begins around P8, a timewhen the retinal vasculature is still rapidly expanding (FIG. 15).Subsequent degeneration of the deep retinal vasculature occurs evenwhile the intermediate plexus is still forming and, thus, the retinas ofrd1/rd1 mice never completely develops, unlike that observed in mosthumans with this disease. An rd10 mouse model, which has a slower timecourse of degeneration and more closely resembles the human retinaldegenerative condition, was used to investigate Lin⁻HSC-mediatedvascular rescue. In the rd10 mouse, photoreceptor cell degenerationbegins around P21 and vascular degeneration begins shortly thereafter.

Since normal neurosensory retinal development is largely complete byP21, the degeneration is observed to start after the retina hascompleted differentiation and in this way is more analogous to humanretinal degenerations than the rd1/rd1 mouse model. Lin⁻HSCs or controlcells from rd10 mice were injected into P6 eyes and the retinas wereevaluated at varying time points. At P21 the retinas from both Lin⁻HSCand control cell-injected eyes appeared normal with complete developmentof all vascular layers and normal development of the INL and ONL (FIG.18, panels (D and H)). At approximately P21 the retinal degenerationbegan and progressed with age. By P30, the control cell-injected retinasexhibited severe vascular and neuronal degeneration (FIG. 18, panel(I)), while the Lin⁻HSC-injected retinas maintained nearly normalvascular layers and photoreceptor cells (FIG. 18, panel (E)). Thedifference between the rescued and non-rescued eyes was more pronouncedat later time points (compare FIG. 18, panels (F and G) to FIG. 18,panels (J and K)). In the control treated eyes, the progression ofvascular degeneration was very clearly observed by immunohistochemicalstaining for CD31 and collagen IV (FIG. 18, panel (I-K)). Thecontrol-treated eyes were nearly completely negative for CD31, whereascollagen IV-positive vascular “tracks” remained evident, indicating thatvascular regression, rather than incomplete vascular formation, hadoccurred. In contrast, Lin⁻HSC-treated eyes had both CD31 and collagenIV-positive vessels that appeared very similar to normal, wild-type eyes(compare FIG. 18, panels (F and I)).

Gene Expression Analysis of rd/rd Mouse Retinas after Lin⁻HSC Treatment.Large scale genomics (microarray analysis) was used to analyze rescuedand non-rescued retinas to identify putative mediators of neurotrophicrescue. Gene expression in rd1/rd1 mouse retinas treated with Lin⁻HSCswas compared to uninjected retinas as well as retinas injected withcontrol cells (CD31⁻). These comparisons each were performed intriplicate. To be considered present, genes were required to haveexpression levels at least 2-fold higher than background levels in allthree triplicates. Genes that were upregulated 3-fold inLin⁻HSC-protected retinas compared to control cell-injected andnon-injected rd/rd mouse retinas are shown in FIG. 20, panels A and B.Coefficient of variance (COV) levels were calculated for the expressedgenes by dividing the standard deviation by the mean expression level ofeach cRNA replicate. In addition, the correlation between expressionlevels and noise variance was calculated by correlating the mean andstandard deviation (SD). A correlation between gene expression level andstandard deviation for each gene was obtained, allowing backgroundlevels and reliable expression level thresholds to be determined. As awhole, the data fell well within acceptable limits (Tu et al. 2002,Proc. Natl. Acad. Sci. USA 99: 14031-14036). The genes that arediscussed individually, below, exhibited expression levels above thesecritical expression levels. Paired “t-test” values for the discussedgenes are also presented in Table 1. In each case, p-values arereasonable (near or below 0.05), which demonstrates that there aresimilarities between replicates and probable significant differencesbetween the different test groups. Many of the significantly upregulatedgenes, including MAD and Ying Yang-1 (YY-1) (Austen et al. 1997, Curr.Top. Microbiol. Immunol. 224: 123-130.), encode proteins with functionsinvolving the protection of cells from apoptosis. A number of crystallingenes, which have sequence homology and similar functions to knownheat-shock proteins involving protection of cells from stress, were alsoupregulated by Lin-HSC treatment. Expression of α-crystallin waslocalized to the ONL by immunohistochemical analysis (FIG. 19). FIG. 19shows that crystallin αA is up regulated in rescued outer nuclear layercells after treatment with Lin⁻HSCs but not in contralateral eyestreated with control cells. The left panel shows IgG staining (control)in rescued retina. The middle panel shows crystallin αA in a rescuedretina. The right panel shows crystallin αA in non-rescued retina.

Messenger RNA from rd1/rd1 mouse retinas rescued with human Lin⁻HSCswere hybridized to human specific Affymetrix U133A microarray chips.After stringent analysis, a number of genes were found whose mRNAexpression was human specific, above background, and significantlyhigher in the human Lin⁻HSC rescued retinas compared to the murineLin⁻HSC rescued retinas and the human control cell-injected non-rescuedretinas (FIG. 20, panel C). CD6, a cell adhesion molecule expressed atthe surface of primitive and newly differentiated CD34+ hematopoieticstem cells, and interferon alpha 13, another gene expressed byhematopoietic stem cells, were both found by the microarraybioinformatics technique, validating the evaluation protocol. Inaddition, several growth factors and neurotrophic factors were expressedabove background by human Lin⁻HSC rescued mouse retina samples (FIG. 20,panel D).

Markers for lineage-committed hematopoietic cells were used tonegatively select a population of bone marrow-derived Lin⁻HSC containingEPC. While the sub-population of bone marrow-derived Lin⁻HSC that canserve as EPC is not characterized by commonly used cell surface markers,the behavior of these cells in developing or injured retinal vasculatureis entirely different than that observed for Lin⁺ or adult endothelialcell populations. These cells selectively target to sites of retinalangiogenesis and participate in the formation of patent blood vessels.

Inherited retinal degenerative diseases are often accompanied by loss ofretinal vasculature. Effective treatment of such diseases requiresrestoration of function as well as maintenance of complex tissuearchitecture. While several recent studies have explored the use ofcell-based delivery of trophic factors or stem cells themselves, somecombination of both may be necessary. For example, use of growth factortherapy to treat retinal degenerative disease resulted in unregulatedovergrowth of blood vessels resulting in severe disruption of the normalretinal tissue architecture. The use of neural or retinal stem cells totreat retinal degenerative disease may reconstitute neuronal function,but a functional vasculature will also be necessary to maintain retinalfunctional integrity. Incorporation of cells from a Lin⁻HSCs of thepresent invention into the retinal vessels of rd/rd mice stabilized thedegenerative vasculature without disrupting retinal structure. Thisrescue effect was also observed when the cells were injected into P15rd/rd mice. Since vascular degeneration begins on P16 in rd/rd mice,this observation expands the therapeutic window for effective Lin⁻HSCtreatment. Retinal neurons and photoreceptors are preserved and visualfunction is maintained in eyes injected with the Lin⁻HSC of the presentinvention.

Adult bone marrow-derived Lin⁻HSCs exert profound vasculo- andneurotrophic effects when injected intravitreally into mice with retinaldegenerative disease. This rescue effect persists for up to 6 monthsafter treatment and is most efficacious when the Lin⁻HSCs are injectedprior to complete retinal degeneration (up to 16 days after birth inmice that ordinarily exhibit complete retinal degeneration by 30 dayspostnatally). This rescue is observed in 2 mouse models of retinaldegeneration and, remarkably, can be accomplished with adult human bonemarrow-derived HSCs when the recipient is an immunodeficient rodent withretinal degeneration (e.g., the SCID mouse) or when the donor is a mousewith retinal degeneration. While several recent reports have described apartial phenotypic rescue in mice or dogs with retinal degenerationafter viral based gene rescue with the wild type gene (Ali, et al. 2000,Nat Genet 25:306-310; Takahashi et al. 1999, J. Virol. 73:7812-7816;Acland et al. 2001, Nat. Genet. 28:92-95.), the present invention is thefirst generic cell-based therapeutic effect achieved by vascular rescue.Thus, the potential utility of such an approach in treating a group ofdiseases (e.g., retinitis pigmentosa) with over 100 known associatedmutations is more practical than creating individual gene therapies totreat each known mutation.

The precise molecular basis of the neurotrophic rescue effect remainsunknown, but is observed only when there is concomitant vascularstabilization/rescue. The presence of injected stem cells, per se, isnot sufficient to generate a neurotrophic rescue and the clear absenceof stem cell-derived neurons in the outer nuclear layer rules out thepossibility that the injected cells are transforming intophotoreceptors. Data obtained by microarray gene expression analysisdemonstrated a significant up-regulation of genes known to haveanti-apoptotic effects. Since most neuronal death observed in retinaldegenerations is by apoptosis, such protection may be of greattherapeutic benefit in prolonging the life of photoreceptors and otherneurons critical to visual function in these diseases. C-myc is atranscription factor that participates in apoptosis by upregulationvarious downstream apoptosis-inducing factors. C-myc expression wasincreased 4.5 fold in rd/rd mice over wild-type indicating potentialinvolvement in the photoreceptor degeneration observed in the rd1/rd1mouse. Mad1 and YY-1, two genes dramatically upregulated inLin⁻HSC-protected retinas (FIG. 20, panel A), are known to suppress theactivity of c-myc, thus inhibiting c-myc induced apoptosis.Overexpression of Mad1 has also been shown to suppress Fas-inducedactivation of caspase-8, another critical component of the apoptoticpathway. Upregulation of these two molecules may play a role inprotection of the retina from vascular and neural degeneration bypreventing the initiation of apoptosis that normally leads todegeneration in rd/rd mice.

Another set of genes that were greatly upregulated in Lin⁻HSC protectedretinas includes members of the crystallin family (FIG. 20, panel B).Similar to heat-shock and other stress-induced proteins, crystallins maybe activated by retinal stress and provide a protective effect againstapoptosis. Abnormally low expression of crystallin αA is correlated withphotoreceptor loss in a rat model of retinal dystrophy and a recentproteomic analysis of the retina in the rd/rd mouse demonstratedinduction of crystalline up-regulation in response to retinaldegeneration. Based on our microarray data of EPC-rescued rd/rd mouseretinas, upregulation of crystallins appear to play a key role in EPCmediated retinal neuroprotection.

Genes such as c-myc, Mad1, Yx-1 and the crystallins are likely to bedownstream mediators of neuronal rescue. Neurotrophic agents canregulate anti-apoptotic gene expression, although our microarrayanalysis of retinas rescued with mouse stem cells did not demonstrateinduction of increased levels of known neurotrophic factors. Analysis ofhuman bone marrow-derived stem cell-mediated rescue with human specificchips did, on the other hand, demonstrate low, but significant increasesin the expression of multiple growth factor genes.

The upregulated genes include several members of the fibroblast growthfactor family and otoferlin. Mutations in the otoferlin gene areassociated with genetic disorders leading to deafness due to auditoryneuropathy. It is possible that otoferlin production by injectedLin⁻HSCs contributes to the prevention of retinal neuropathy as well.Historically, it has long been assumed that vascular changes observed inpatients and animals with retinal degeneration were secondary todecreased metabolic demand as the photoreceptors die. The present dataindicate that, at least for mice with inherited retinal degeneration,preserving normal vasculature can help maintain components of the outernuclear layer as well. Recent reports in the literature would supportthe concept that tissue-specific vasculature has trophic effects that gobeyond that expected from simply providing vascular “nourishment.” Forexample, liver endothelial cells can be induced to produce, after VEGFR1activation, growth factors critical to hepatocyte regeneration andmaintenance in the face of hepatic injury (LeCouter et al. 2003, Science299:890-893).

Similar indicative interactions between vascular endothelial cells andadjacent hepatic parenchymal cells are reportedly involved in liverorganogenesis, well before the formation of functional blood vessels.Endogenous retinal vasculature in individuals with retinal degenerationmay not facilitate so dramatic a rescue, but if this vasculature isbuttressed with endothelial progenitors derived from bone marrowhematopoietic stem cell populations, they may make the vasculature moreresistant to degeneration and at the same time facilitate retinalneuronal, as well as vascular, survival. In humans with retinaldegeneration, delaying the onset of complete retinal degeneration mayprovide years of additional sight. The animals treated with the Lin⁻HSCsof the present invention had significant preservation of an ERG, whichmay be sufficient to support vision.

Clinically, it is widely appreciated that there may be substantial lossof photoreceptors and other neurons while still preserving functionalvision. At some point, the critical threshold is crossed and vision islost. Since nearly all of the human inherited retinal degenerations areof early, but slow, onset, an individual with retinal degeneration canbe identified and treated intravitreally with a graft of autologous bonemarrow stem cells of the invention to delay retinal degeneration andconcomitant loss of vision. To enhance targeting and incorporation ofthe stem cells of the invention, the presence of activated astrocytes isdesirable (Otani et al. 2002, Nat. Med. 8: 1004-1010); this can beaccomplished by early treatment when there is an associated gliosis, orby using a laser to stimulate local proliferation of activatedastrocytes. Optionally, ex vivo transfection of the stem cells with oneor more neurotrophic substances prior to intraocular injection can beused to enhance the rescue effect. This approach can be applied to thetreatment of other visual neuronal degenerative disorders, such asglaucoma, in which there is retinal ganglion cell degeneration.

The Lin⁻HSC populations of the present invention contain a population ofEPC that can promote angiogenesis by targeting reactive astrocytes andincorporate into an established template without disrupting retinalstructure. The Lin⁻HSC of the present invention also provide asurprising long-term neurotrophic rescue effect in eyes suffering fromretinal degeneration. In addition, genetically modified, autologousLin⁻HSC compositions containing EPC can be transplanted into ischemic orabnormally vascularized eyes and can stably incorporate into new vesselsand neuronal layers and continuously deliver therapeutic moleculeslocally for prolonged periods of time. Such local delivery of genes thatexpress pharmacological agents in physiologically meaningful dosesrepresents a new paradigm for treating currently untreatable oculardiseases.

Photoreceptors in the normal mouse retina, for example, arepredominantly rods, but the outer nuclear layer observed after rescuewith Lin-HSCs of the invention contained predominantly cones. Mostinherited human retinal degenerations occur as a result of primaryrod-specific defects, and loss of the cones is believed to be secondaryto rod dysfunction, which is likely related to the loss of some trophicfactor expressed by rods. The present method of inducing cone survivalin the face of rod/retinal degeneration facilitated by Lin⁻HSC, affordsa way to better preserve the cone-dominated human macula in diseasessuch as retinitis pigmentosa.

Numerous variations and modifications of the embodiments described abovemay be effected without departing from the spirit and scope of the novelfeatures of the invention. No limitations with respect to the specificembodiments illustrated herein are intended or should be inferred.

1. A method of preserving cone cells in the eye of a mammal sufferingfrom a retinal degenerative disease comprising isolating from the bonemarrow of the mammal a lineage negative hematopoietic stem cellpopulation that includes endothelial progenitor cells, transfectingcells from the stem cell population with a gene that operably encodes anantiangiogenic fragment of human tryptophanyl tRNA synthetase (TrpRS),and subsequently intravitreally injecting the transfected cells into theeye of the mammal in an amount sufficient to inhibit the degeneration ofcone cells in the retina of the eye.
 2. The method of claim 1 wherein atleast about 75% of the total cells of the stem cell population expressCD31.
 3. The method of claim 1 wherein at least about 50% of the totalcells of the stem cell population express integrin α₆.
 4. The method ofclaim 1 wherein the stem cell population comprises adult bone marrowcells.
 5. The method of claim 1 wherein the mammal is a mouse.
 6. Themethod of claim 5 wherein at least about 80% of the total cells of thestem cell population express CD31 and at least about 70% of the totalcells of the stem cell population express CD117.
 7. The method of claim1 wherein the mammal is a human.
 8. The method of claim 7 wherein thecells in the stem cell population are CD133 negative, at least about 50%of the total cells of the stem cell population express integrin α₆, andat least about 50% of the total cells of the stem cell populationexpress CD31.
 9. The method of claim 7 wherein the cells in the stemcell population are CD133 positive, less than about 30% of the totalcells of the stem cell population express integrin α₆, and less thanabout 30% of the total cells of the stem cell population express CD31.10. The method of claim 1 wherein the TrpRS fragment is selected fromthe group consisting of T2-TrpRS (SEQ ID NO: 3) and T2-TrpRS-GD (SEQ IDNO: 4).
 11. The method of claim 1 further comprising transfecting thecells with a gene that operably encodes a therapeutically usefulpeptide, in addition to the TrpRS fragment, prior to injecting the cellsinto the eye.
 12. The method of claim 11 wherein the therapeuticallyuseful peptide is an anti-angiogenic peptide.
 13. The method of claim 11wherein the therapeutically useful peptide is a neurotrophic agent. 14.The method of claim 13 wherein the neurotrophic agent is selected formthe group consisting of nerve growth factor, neurotrophin-3,neurotrophin-4, neurotrophin-5, ciliary neurotrophic factor, retinalpigmented epithelium-derived neurotrophic factor, insulin-like growthfactor, glial cell line-derived neurotrophic factor, and brain-derivedneurotrophic factor.
 15. The method of claim 1 wherein astrocytes in theretina are stimulated using a laser prior to injecting the cells intothe eye.
 16. A method of preserving cone cells in the eye of a mammalsuffering from a retinal degenerative disease comprising intravitreallyinjecting cells from a transfected autologous stem cell population intothe eye of the mammal in an amount sufficient to ameliorate thedegeneration of cone cells in the retina thereof, wherein the autologousstem cell population has been transfected with a nucleic acid capable ofexpressing an antiangiogenic fragment of human tryptophanyl tRNAsynthetase (TrpRS); and wherein the fragment of TrpRS is selected fromthe group consisting of T2-TrpRS (SEQ ID NO: 3) and T2-TrpRS-GD (SEQ IDNO: 4).
 17. The method of claim 16 wherein astrocytes in the retina ofthe mammal are stimulated using a laser prior to intravitreallyinjecting the cells.
 18. The method of claim 16 wherein thelineage-negative hematopoietic stem cell population has been isolatedby: (a) extracting bone marrow from the mammal; (b) separating aplurality of monocytes from the bone marrow; (c) labeling the pluralityof monocytes with biotin-conjugated lineage panel antibodies to one ormore lineage surface antigens; and (d) separating monocytes that arepositive for said one or more lineage surface antigens from theplurality of monocytes and recovering a population of lineage negativehematopoietic stem cells containing endothelial progenitor cells. 19.The method of claim 18 wherein the one or more lineage surface antigensare selected from the group consisting of CD2, CD3, CD4, CD11, CD11a,Mac-1, CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, Ly-6G, TER-119,CD45RA, CD56, CD64, CD68, CD86, CD66b, HLA-DR, and CD235a.
 20. A methodof preserving cone cells in the retina of a mammal suffering from aretinal degenerative disease comprising isolating from the bone marrowof the mammal a lineage negative hematopoietic stem cell population thatincludes endothelial progenitor cells, transfecting the isolated cellswith a gene that operably encodes an antiangiogenic fragment of humantryptophanyl tRNA synthetase, treating the retina with a laser tostimulate local proliferation of activated astrocytes in the retina, andsubsequently intravitreally injecting the transfected stem cells intothe eye of the mammal in an amount sufficient to ameliorate thedegeneration of cone cells in the retina.